Protocol for the Process Evaluation of the Online Remote Behavioural Intervention for Tics (ORBIT) randomized controlled trial for children and young people.

BACKGROUND: Process evaluations are an important component in the interpretation and understanding of outcomes in trials. The Online Remote Behavioural Intervention for Tics (ORBIT) study is a randomized controlled trial evaluating the effectiveness of an Internet-delivered behavioural intervention (called BIP TIC) compared to an Internet-delivered education programme aimed at children and young people with tics. A process evaluation will be undertaken alongside the main trial to determine precisely how the behavioural intervention works and ascertain whether, and if so, how, the intervention could be successfully implemented in standard clinical practice. This protocol paper describes the rationale, aims, and methodology of the ORBIT trial process evaluation. METHODS: The process evaluation will have a mixed-methods design following the UK Medical Research Council 2015 guidelines, comprising both quantitative and qualitative data collection. This will include analysing data usage of participants in the intervention arm; purposively sampled, semi-structured interviews of parents and children, therapists and supervisors, and referring clinicians of the ORBIT trial, as well as analysis of qualitative comments put into the online therapy platform by participants at the end of treatment. Qualitative data will be analysed thematically. Quantitative and qualitative data will be integrated in a triangulation approach, to provide an understanding of how the intervention works, and what resources are needed for effective implementation, uptake and use in routine clinical care. DISCUSSION: This process evaluation will explore the experiences of participants, therapists and supervisors and referring clinicians of a complex online intervention. By contextualising trial efficacy results, this will help understand how and if the intervention worked and what may be required to sustain the implementation of the treatment long term. The findings will also aid in our understanding of factors that can affect the success of complex interventions. This will enable future researchers developing online behavioural interventions for children and young people with mental health and neurological disorders to gain invaluable information from this process evaluation. TRIAL REGISTRATION: International Standard Randomised Controlled Trials Number, ISRCTN70758207. Registered on 20 March 2018. ClinicalTrials.gov, NCT03483493. Registered on 30 March 2018.

Future technology insight: mass spectrometry imaging as a tool in drug research and development.

In pharmaceutical research, understanding the biodistribution, accumulation and metabolism of drugs in tissue plays a key role during drug discovery and development. In particular, information regarding pharmacokinetics, pharmacodynamics and transport properties of compounds in tissues is crucial during early screening. Historically, the abundance and distribution of drugs have been assessed by well-established techniques such as quantitative whole-body autoradiography (WBA) or tissue homogenization with LC/MS analysis. However, WBA does not distinguish active drug from its metabolites and LC/MS, while highly sensitive, does not report spatial distribution. Mass spectrometry imaging (MSI) can discriminate drug and its metabolites and endogenous compounds, while simultaneously reporting their distribution. MSI data are influencing drug development and currently used in investigational studies in areas such as compound toxicity. In in vivo studies MSI results may soon be used to support new drug regulatory applications, although clinical trial MSI data will take longer to be validated for incorporation into submissions. We review the current and future applications of MSI, focussing on applications for drug discovery and development, with examples to highlight the impact of this promising technique in early drug screening. Recent sample preparation and analysis methods that enable effective MSI, including quantitative analysis of drugs from tissue sections will be summarized and key aspects of methodological protocols to increase the effectiveness of MSI analysis for previously undetectable targets addressed. These examples highlight how MSI has become a powerful tool in drug research and development and offers great potential in streamlining the drug discovery process.

Internet-based cognitive behaviour therapy for obsessive-compulsive disorder: a randomized controlled trial.

BACKGROUND: Cognitive behaviour therapy (CBT) is an effective treatment for obsessive-compulsive disorder (OCD) but access to CBT is limited. Internet-based CBT (ICBT) with therapist support is potentially a more accessible treatment. There are no randomized controlled trials testing ICBT for OCD. The aim of this study was to investigate the efficacy of ICBT for OCD in a randomized controlled trial. METHOD: Participants (n=101) diagnosed with OCD were randomized to either 10 weeks of ICBT or to an attention control condition, consisting of online supportive therapy. The primary outcome measure was the Yale-Brown Obsessive Compulsive Scale (YBOCS) administered by blinded assessors. RESULTS: Both treatments lead to significant improvements in OCD symptoms, but ICBT resulted in larger improvements than the control condition on the YBOCS, with a significant between-group effect size (Cohen's d) of 1.12 (95% CI 0.69-1.53) at post-treatment. The proportion of participants showing clinically significant improvement was 60% (95% CI 46-72) in the ICBT group compared to 6% (95% CI 1-17) in the control condition. The results were sustained at follow-up. CONCLUSIONS: ICBT is an efficacious treatment for OCD that could substantially increase access to CBT for OCD patients. Replication studies are warranted.

Neuropeptidomics: expanding proteomics downwards.

Biological function is mainly carried out by a dynamic population of proteins and peptides which may be used as markers for disease diagnosis, prognosis and as a guide for effective treatment. The study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel-based proteomics is methodologically restricted from the low mass region, which includes important endogenous peptides. The study of endogenous peptides, peptidomics, is complicated by protein fragments produced post-mortem during conventional sample handling. Nanoflow liquid chromatography and MS, together with improved methods for sample preparation, have been used to semi-quantitatively monitor endogenous peptides in brain tissue. When rapidly heat-denatured brain tissue was analysed, these methods enabled simultaneous detection of hundreds of peptides and the identification of several endogenous peptides not previously described in the literature. In an application of the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model for Parkinson's disease, the expression of the small protein PEP-19 was compared with controls. The levels were found to be significantly decreased in the striatum of MPTP-treated animals.

Naloxone reduces levodopa-induced dyskinesias and apomorphine-induced rotations in primate models of parkinsonism.

Using in situ hybridization, it was found that subchronic treatment with levodopa/benserazide increased preproenkephalin-A and preproenkephalin-B mRNAs in the dopamine-depleted striatum. In order to examine whether dysfunction of the endogenous opioid system may underlie the development of levodopa-induced dyskinesias, the effect of naloxone, an opioid antagonist, on dyskinesias was investigated in two models of parkinsonism in the common marmoset. MPTP-treated monkeys were administered a daily oral dose of levodopa/benserazide which relieved the parkinsonian symptoms but induced severe and reproducible dyskinetic movements. Naloxone (0.1, 0.2 or 0.5 mg/kg) was given subcutaneously (s.c.) during peak-dose dyskinesia, which reduced the dyskinesias significantly using the highest dose, normalized the motor activity, but did not modify the antiparkinson effect. Unilaterally 6-OHDA -lesioned marmosets received apomorphine s.c., which caused a contralateral turning behavior that could be reduced up to 35 percent by concomitant administration of naloxone. Taken together the present results suggest a possible role for the endogenous opioid system in the pathogenesis of levodopa-induced dyskinesia in primates.

Coadministration of (-)-OSU6162 with l-DOPA normalizes preproenkephalin mRNA expression in the sensorimotor striatum of primates with unilateral 6-OHDA lesions.

The substituted phenylpiperidine (-)-OSU6162 is a novel modulator of the dopaminergic systems with low affinity for dopamine D(2) receptors and potent normalizing effects on l-DOPA-induced dyskinesias. We studied the effects of coadministration of (-)-OSU6162 with l-DOPA on the regulation of striatal preproenkephalin (PPE) and prodynorphin (PDyn) mRNA expression in the primate brain by in situ hybridization histochemistry. Common marmoset monkeys sustaining unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received l-DOPA/carbidopa, l-DOPA/carbidopa plus (-)-OSU6162, or vehicle over 14 days. In vehicle-treated animals, PPE mRNA levels were markedly increased in the sensorimotor territory of the lesioned striatum. By contrast, a rather uniform lesion-induced reduction of PDyn mRNA levels was found in the vehicle group. Subchronic l-DOPA treatment induced a further increase in PPE mRNA expression in a number of sensorimotor and associative subregions of the denervated striatum. Coadministration of (-)-OSU6162 with l-DOPA partially reversed the lesion- and l-DOPA-induced elevation of PPE expression and, by affecting PPE mRNA expression differentially on the intact and lesioned striatum, markedly reduced the side-to-side difference in PPE mRNA expression. The effects on PPE mRNA expression were apparent throughout the rostrocaudal extent of the putamen and the dorsal portions of the caudate nucleus. l-DOPA treatment resulted in an enhancement in PDyn mRNA expression in all functional compartments of the striatum. Coadministration of (-)-OSU6162 had no apparent influence on these l-DOPA-induced changes in PDyn mRNA expression. The present results suggest that (-)-OSU6162 acts primarily by modifying striatal output via the indirect pathway.

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers.

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.

L-DOPA produces strong induction of c-fos messenger RNA in dopamine-denervated cortical and striatal areas of the common marmoset.

Common marmosets (Callithrix jacchus) with near-complete unilateral 6-hydroxydopamine denervation of the dopaminergic input received a single injection of saline or L-DOPA (15mg/kg plus 6.25mg/kg benserazide). Using in situ hybridization, the effects of these treatments on c-fos messenger RNA expression in the cerebral cortex, the striatal complex and the external layer of the pallidum were studied. Moreover, receptor autoradiography was used to determine the levels of dopamine D(1) and D(2) receptors in these areas. In the cerebral cortex, animals treated with L-DOPA displayed a high expression of c-fos messenger RNA restricted to the dopamine-denervated hemisphere. No changes in the levels of cortical D(1) and D(2) receptors were found in the dopamine-denervated hemisphere. L-DOPA treatment also induced a strong expression of c-fos messenger RNA in the striatal complex in the dopamine-denervated hemisphere. The levels of striatal D(2), but not D(1), receptors were increased in the dopamine-denervated hemisphere. In the external pallidum, the major terminal region for D(2) dopamine receptor-containing striatal projection neurons, L-DOPA treatment induced c-fos messenger RNA expression in both the intact and the dopamine-denervated hemispheres.Thus, using c-fos messenger RNA as a biochemical marker of postsynaptic neuronal activation, these results provide evidence that near-complete dopamine depletion causes a profound supersensitization to L-DOPA treatment in the cerebral cortex and in the striatal complex, but not in the external layer of the pallidum, of the primate brain. The cortical response may be unique to the primate brain, but c-fos messenger RNA activation within the striatum has also been reported in the rodent. The effects of L-DOPA probably depend both on a direct activation of supersensitized dopamine receptors by dopamine produced in the few remaining, but hyperactive, dopaminergic nerve terminals and in serotonergic nerve terminals, as well as on indirect actions of L-DOPA related to activation of circuitries connecting cerebral cortex and basal ganglia structures. These results provide novel information on the mechanisms underlying L-DOPA's action in the cerebral cortex, striatum and external pallidum in a primate model of Parkinson's disease.

Motor effects of (-)-OSU6162 in primates with unilateral 6-hydroxydopamine lesions.

The effects of the novel compound, (-)-OSU6162 ((S)-(-)-3-methylsulfonylphenyl-1-propylpiperidine), on rotational behavior induced by dopamine receptor agonists was investigated in common marmosets (Callithrix jacchus) with unilateral 6-hydroxydopamine lesions. (-)-OSU6162 per se displayed no effect on the animals' behavior. On the other hand, pretreatment with (-)-OSU6162 attenuated rotational behavior induced by apomorphine (apomorphini hydrochloridum), L-DOPA (3,4-dihydroxyphenylalanine), and the dopamine D2 receptor agonist, quinpirole (trans-(-)-4aR-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolol[3,4-g]quinoline hydrochloride), without inducing motor impairment such as akinesia or dystonia. In addition, treatment with (-)-OSU6162 for 5 consecutive days almost completely abolished the rotational behavior provoked by apomorphine and produced a transient subsensitization of such apomorphine-induced effects after it was discontinued. Moreover, pretreatment with (-)-OSU6162 in two monkeys augmented the rotational behavior elicited by the dopamine D1 receptor agonists, SKF-81297 (R(+)-6-chloro-7,8,dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine hydrobromide) and A-77636 ((-)-(1R, 3S)-3-adamantyl-1-(aminomethyl)-3,4-dihydro-5, 6-dihydroxy-1H-2-benzopyran hydrochloride). The findings indicate that (-)-OSU6162 can exert indirect state-dependent effects that differentially affect dopamine D1 and dopamine D2 receptor agonist-induced behavior.

Determination of extracellular release of neurotensin in discrete rat brain regions utilizing in vivo microdialysis/electrospray mass spectrometry.

In vivo microdialysis was used together with structure-specific high sensitivity nano-flow capillary liquid chromatography/micro-electrospray mass spectrometry to quantify and compare extracellular neurotensin from discrete regions of the rat brain. Microdialysis probes were implanted in the hypothalamus or globus pallidus/ventral pallidum in unanesthetized freely moving animals. Utilizing this specific methodology, recovered basal levels of neurotensin were detectable in hypothalamus and globus pallidus/ventral pallidum. The basal level of neurotensin in these regions were slightly higher in hypothalamus (101+/-11 amol/10 microl, n=6) compared to those in the globus pallidus/ventral pallidum region (74+/-12 amol/10 microl, n=8) in samples collected for 30 min at a flow-rate of 0.4 microl/min 150-180 min after the microdialysis probe implantation. After a pulse of 1.0 microl of 100 mM KCl-containing artificial cerebrospinal fluid during the next 30-min sampling period (180-210 min), the recovered neurotensin increased in hypothalamus and globus pallidus/ventral pallidum by 544% (548+/-90 amol/10 microl) and 674% (499+/-99 amol/10 microl), respectively. The basal levels of endogenously released neurotensin in the hypothalamus and globus pallidus/ventral pallidum were lower in the present study compared to those previously reported in the rat brain using in vivo microdialysis and radioimmunoassays. Our data demonstrate the effectiveness of combining in vivo microdialysis and structure-specific micro-electrospray mass spectrometry for the quantitation of basal and stimulated in vivo levels of endogenous neurotensin (NT) in different brain areas.

Simultaneous analysis of endogenous neurotransmitters and neuropeptides in brain tissue using capillary electrophoresis--microelectrospray-tandem mass spectrometry.

Capillary electrophoresis was combined with highly sensitive microelectrospray-tandem mass spectrometry to simultaneously detect classical small molecule neurotransmitters as well as neuropeptides from discrete regions of the marmoset brain. A mixture of four classical neurotransmitters (glutamate, gamma-aminobutyric acid, acetylcholine, dopamine) and four neuropeptides (neurotensin, methionine-enkephalin, leucine-enkephalin and substance P 1-7) was studied to optimize the capillary electrophoresis conditions for separation, injection volume, and analysis time. Gamma-aminopropyltriethoxysilane-coated capillaries and acetic acid electrolytes were used to avoid interactions between the sample and the capillary surface and to obtain a high anodic electroosmotic flow, which resulted in a short analysis time. Detection was performed using tandem mass spectrometry in the selected reaction monitoring mode using a triple quadrupole mass spectrometer. Samples were dissolved in ammonium acetate to achieve a transient-isotachophoretic concentration step at the beginning of the separation and to make it possible to inject larger sample volumes, up to 140 nL. Small amounts of tissue from specific regions of the marmoset monkey brain were pretreated using solid-phase extraction as a clean-up and concentrating step. In the striatum we could detect endogenous glutamate, gamma-aminobutyric acid (GABA), acetylcholine and dopamine, as well as the neuropeptides methionine-enkephalin and substance P 1-7 in the same analysis, using only 58 mm3 of brain tissue.

Combining solid-phase preconcentration, capillary electrophoresis and off-line matrix-assisted laser desorption/ionization mass spectrometry: intracerebral metabolic processing of peptide E in vivo.

The in vivo metabolism of peptide E was studied in the anesthetized rat using a combination of microdialysis sampling, solid-phase preconcentration capillary electrophoresis and imaging matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). The metabolic profile of peptides identified by MALDI/MS showed that the primary enzymatic activity for degradation of peptide E was due to carboxypeptidases and, to a lesser extent, aminopeptidases and some trypsin-like endopeptidases. Over 75 metabolic fragments were detected from the action of these enzymes in vivo.

Blood-brain barrier penetration of 3-aminopropyl-n-butylphosphinic acid (CGP 36742) in rat brain by microdialysis/mass spectrometry.

The detection and quantitation of the novel drug 3-aminopropyl-n-butylphosphinic acid (APBP), also known as CGP 36742, was performed in vivo using microdialysis and tandem mass spectrometry. This drug is a GABA-B antagonist with high specificity for GABA-B receptors. Animals received doses of 100, 200, 500 and 1000 mg kg-1 of the drug either intravenously or per os (p.o.). Microdialysis probes, placed by stereotaxis in either the frontal cortex or third ventricle of the rat, were used to collect dialyzate samples over several hours. Samples were then analyzed by micro-electrospray tandem mass spectrometry to achieve a molecular mass and structure specific analysis. For example, animals receiving a dose of 100 mg kg-1 p.o. showed a peak concentration of approximately 10 microM in the dialyzate. For comparison, tissue and plasma samples of the drug were measured under the same conditions using gas chromatography/mass spectrometry. This work demonstrates that the microdialysis technique in combination with the molecular specificity and high sensitivity of micro-electrospray tandem mass spectrometry can be used to study the time course of the appearance of unmodified drug in the brain of a single animal.

(-)-OSU 6162 inhibits levodopa-induced dyskinesias in a monkey model of Parkinson's disease.

We have studied the effects of two D2 dopamine receptor-selective compounds, (-)-OSU 6162 and raclopride, on levodopa-induced dyskinesias in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned common marmosets (Callithrix jacchus). Three monkeys developed a severe parkinsonian syndrome following administration of MPTP. In response to daily levodopa treatment the animals developed reproducible and idiosyncratic peak-dose dyskinesias. Pretreatment with (-)-OSU 6162 and raclopride, in doses increased by multiples of three, both dose-dependently relieved the levodopa-induced dyskinesias. However, in contrast to when raclopride pretreatment was given, (-)-OSU 6162 pretreatment did not induce akinesia. Our investigation suggests that (-)-OSU 6162 may be useful an an adjuvant treatment to levodopa in advanced Parkinson's disease to selectively combat levodopa-induced dyskinesias without affecting the antiparkinsonian response.

Purification and characterization of substance P endopeptidase activities in the rat spinal cord.

Two enzymes with substance P degrading activity were purified from the membrane bound fraction of the rat spinal cord. The purified enzymes were characterized with regard to biochemical and kinetic properties. One of the enzymes exhibited close similarity to neutral endopeptidase 24.11 (NEP, EC 3.4.24.11), while the other resembled a substance P converting endopeptidase (SPE), which has previously been identified and purified from human cerebrospinal fluid (CSF). Detergent treated spinal cord homogenates from male Sprague Dawley rats were purified by anion-exchange chromatography (DEAE-sepharose CL-6B), hydrophobic-interaction chromatography (phenyl-sepharose CL-4B) and molecular sieving (Sephadex G-50). Two fractions with enzymes differing in size were recovered and allowed for further purification to apparent homogeneity by ion-exchange chromatography and molecular sieving on a micro-purification system (SMART). The enzyme activities were monitored by following the conversion of synthetic substance P using a radioimmunoassay specific for the heptapeptide product, substance P (1-7). By SDS-polyacrylamide gel electrophoresis of the purified enzymes molecular weights of 43 and 70 kDa were estimated for the SPE-like and NEP-like activity, respectively. A K(m) of 5 microM was determined for the conversion of substance P to its (1-7) fragment by the SPE-like activity. Reversed-phase HPLC together with mass spectrometry permitted identification of all fragments released from substance P by the peptidases. The released fragments were for both enzymes identified as substance P (1-7), substance P (8-11), substance P (1-8), substance P (9-11). The NEP-like enzyme preparation also gave substance P (1-6) as a major product.

In vitro metabolism of LVV-hemorphin-7 in human plasma studied by reversed-phase high-performance liquid chromatography and micro-electrospray mass spectrometry.

In these experiments we have studied the in vitro metabolism of LVV-hemorphin-7 in human plasma by using reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with micro-electrospray mass spectrometry (micro-ES-MS). Tandem mass spectrometry (MS-MS) was performed in order to verify the structure of the peptide fragments found. Incubations were performed with and without different protease inhibitors. Results showed that LVV-hemorphin-7 was metabolized from the N-terminal end of the peptide, probably by an amastatin-sensitive exopeptidase.

Reversed-phase high-performance liquid chromatography combined with tandem mass spectrometry in studies of a substance P-converting enzyme from human cerebrospinal fluid.

The application of reversed-phase HPLC in combination with micro-electrospray mass spectrometry to study a substance P (SP)-hydrolysing endoprotease in human cerebrospinal fluid (hCSF) is reported. The enzyme was partially purified from the hCSF specimens by ion-exchange chromatography and molecular sieving. During the purification procedure the enzyme activity was monitored by measuring the formation of the SP-fragment 1-7 from SP by radioimmunoassay. Regarding its behaviour upon molecular sieve chromatography, the enzyme was suggested to be associated with an apparent molecular mass of around 100 x 10(3). In subsequent experiments using the partially purified endopeptidase, the hydrolysis of SP was demonstrated by HPLC. The reaction product mixture was resolved in several components including the N-terminal fragments 1-8, 1-7 and 1-6 and the C-terminal fragment 8-11. The identity of these fragments were confirmed by tandem mass spectrometry. It was concluded that the present SP-degrading enzyme is different from those previously identified and purified from hCSF. The applied techniques were proven to be highly efficient for the recovery and identification of the released peptide products.

Isolation of haemorphin-related peptides from filter membranes collected in connection with haemofiltration of human subjects.

This paper describes the extraction and isolation from dialysis filters of two peptides containing the opioid active sequence haemorphin-7. The filter devices were obtained from uraemic patients subjected to haemofiltration. Following acidic extraction of the filter membranes the peptides were purified by size-exclusion, ion-exchange chromatography and finally by reversed-phase chromatography using different columns and different chromatographic systems. The purification was guided by radioimmunoassay and the structure of the final products was elucidated by N-terminal sequencing and fast-atom bombardment mass spectrometry as well as micro-electrospray mass spectrometry. The isolated peptides were suggested to be identical to fragments 1-41 and 32-41 of the beta-chain of human haemoglobin.

High frequency oral movements induced by long-term administration of amperozide but not FG5803 in rats.

Long-term studies of antipsychotic-induced oral movements may serve as a rat model of acute and tardive movement disorders. Vacuous chewing movements (VCM), tongue protrusions (TP), and jaw tremors (TR) were studied in rats during acute and chronic administration of two potential antipsychotics, amperozide and FG5803. Comparisons were made with haloperidol and vehicle. Single intraperitoneal injections of amperozide (0.2, 1, or 5 mg/kg) or FG5803 (1.2, 6, or 30 mg/kg) were without effect on oral behaviors. During long-term drug administration, withdrawal and readministration, endpoint analysis was focused on changes in supranormal oral movements. The maximal mean control frequencies found at 29 sessions during 14 months experiment +2 standard deviations were used to define the upper limit of the normal range. FG5803 (1.2, 6, or 30 mg/kg per day) administered via the drinking water for 12 months, did not produce significant deviations from this normal range with respect to VCM, TP, or TR, and this drug was not studied further. Rats receiving amperozide (0.2, 1, or 5 mg/kg per day) showed dose-related increases in oral movements over the year. The changes began after 3 months of treatment with amperozide 1 and 5 mg/kg per day, but became statistically significant only during the second half of the treatment year. Amperozide 0.2 mg/kg per day did not produce significant changes in oral movements during administration for a year, but drug withdrawal resulted in a significant rise in TP behavior. Haloperidol (1 mg/kg per day) produced increases in supranormal oral movements which tended to level out after 9 months. In all groups with significant elevations (i.e. haloperidol and amperozide 1 and 5 mg/kg per day), there was a persistence of such movements during a month of drug withdrawal. During treatment with amperozide (1 or 5 mg/kg per day), some rats developed a high frequency chewing behavior up to 175 VCMs/min. It is concluded that long-term treatment with amperozide, but not FG5803, produced a tardive pattern of supranormal oral movements. The importance of these findings for the clinical future of amperozide is difficult to predict, due to the unexpected finding of high-frequency chewing, which has not been noticed before during extensive studies of classical neuroleptics.