Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

We investigated the protective potential of recombinant his-tagged antigens recNcMIC1, recNcMIC3 and recNcROP2, applied either as single vaccines or as vaccine combinations, in BALB/c mouse models for cerebral and fetal infection. Subsequently, mice were mated and challenged by i.p. inoculation of 2 x 10(6)Neospora caninum tachyzoites at day 7 of pregnancy. The mortality and morbidity of adult mice (non-pregnant and dams) and of the newborn pups was studied for a period of 40 days following birth. Vaccination of non-pregnant mice with recNcROP2 or combinations of recNcROP2 with recNcMIC antigens significantly reduced the numbers of mice suffering from clinical signs, and morbidity was completely prevented with the combination of all three antigens. Of the dams, the groups receiving either recNcROP2 alone or the combination of all three antigens did not exhibit any morbidity, the groups receiving ROP2 mixed with either MIC1 or MIC3 exhibited reduced numbers of deaths, and in the infection control group and the adjuvant group 50% and 43% of mice, respectively, succumbed to disease. For pups, the highest survival rates were noted for the groups receiving recNcROP2 (50%) and recNcROP2/NcMIC1/NcMIC3 (35%), while in the infection- and adjuvant- control groups all pups died, the latest at days 25 and 30, respectively. Quantification of parasite DNA by N. caninum-specific real-time PCR revealed consistently lower parasite burdens in brain tissue of pups from vaccinated groups compared with the controls. However, dense granule antigen 2 (GRA2) real-time reverse transcriptase-PCR on brain tissue of surviving pups (applied here to detect viable parasites) demonstrated that only the pups from the group vaccinated with all three antigens in combination appeared free of viable tachyzoites, while in all other groups viable parasites were still present. Serological analysis of humoral (total IgG, IgG1 and IgG2a) and serum cytokine (IL-4 and IFN-gamma) responses showed that this effect was associated with a Th-2-biased immune response, with a clearly elevated IL-4/IFN-gamma ratio in the mice receiving all three antigens in combination. In conclusion, a mixture of recombinant antigens representing important secretory micronemal and rhoptry proteins leads to a significant protection against vertical transmission of N. caninum in mice.

IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology

BACKGROUND: Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the Brucella-specific insertion sequence IS711 with blood samples from 199 wild boars (first group of animals) and tissue samples from 53 wild boars (second group of animals) collected in Switzerland. Results from IS711 real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and indirect ELISA (i-ELISA). RESULTS: In the first group of animals, IS711 real-time PCR detected infection in 11.1% (16/144) of wild boars that were serologically negative. Serological tests showed different sensitivities [RBT 15.6%, c-ELISA 7.5% and i-ELISA 5.5%] and only 2% of blood samples were positive with all three tests, which makes interpretation of the serological results very difficult. Regarding the second group of animals, the IS711 real-time PCR detected infection in 26% of animals, while Brucella spp. could be isolated from tissues of only 9.4% of the animals. CONCLUSION: The results presented here indicate that IS711 real-time PCR assay is a specific and sensitive tool for detection of Brucella spp. infections in wild boars. For this reason, we propose the employment of IS711 real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.

The state of in vitro science for use in bioaccumulation assessments for fish

Through the concerted evaluations of thousands of commercial substances for the qualities of persistence, bioaccumulation, and toxicity as a result of the United Nations Environment Program's Stockholm Convention, it has become apparent that fewer empirical data are available on bioaccumulation than other endpoints and that bioaccumulation models were not designed to accommodate all chemical classes. Due to the number of chemicals that may require further assessment, in vivo testing is cost prohibitive and discouraged due to the large number of animals needed. Although in vitro systems are less developed and characterized for fish, multiple high-throughput in vitro assays have been used to explore the dietary uptake and elimination of pharmaceuticals and other xenobiotics by mammals. While similar processes determine bioaccumulation in mammalian species, a review of methods to measure chemical bioavailability in fish screening systems, such as chemical biotransformation or metabolism in tissue slices, perfused tissues, fish embryos, primary and immortalized cell lines, and subcellular fractions, suggest quantitative and qualitative differences between fish and mammals exist. Using in vitro data in assessments for whole organisms or populations requires certain considerations and assumptions to scale data from a test tube to a fish, and across fish species. Also, different models may incorporate the predominant site of metabolism, such as the liver, and significant presystemic metabolism by the gill or gastrointestinal system to help accurately convert in vitro data into representative whole-animal metabolism and subsequent bioaccumulation potential. The development of animal alternative tests for fish bioaccumulation assessment is framed in the context of in vitro data requirements for regulatory assessments in Europe and Canada.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Male reproductive pattern in a polygynous ungulate with a slow life-history: the role of age, social status and alternative mating tactics

According to life-history theory age-dependent investments into reproduction are thought to co-vary with survival and growth of animals. In polygynous species, in which size is an important determinant of reproductive success, male reproduction via alternative mating tactics at young age are consequently expected to be the less frequent in species with higher survival. We tested this hypothesis in male Alpine ibex (Capra ibex), a highly sexually dimorphic mountain ungulate whose males have been reported to exhibit extremely high adult survival rates. Using data from two offspring cohorts in a population in the Swiss Alps, the effects of age, dominance and mating tactic on the likelihood of paternity were inferred within a Bayesian framework. In accordance with our hypothesis, reproductive success in male Alpine ibex was heavily biased towards older, dominant males that monopolized access to receptive females by adopting the 'tending' tactic, while success among young, subordinate males via the sneaking tactic 'coursing' was in general low and rare. In addition, we detected a high reproductive skew in male Alpine ibex, suggesting a large opportunity for selection. Compared with other ungulates with higher mortality rates, reproduction among young male Alpine ibex was much lower and more sporadic. Consistent with that, further examinations on the species level indicated that in polygynous ungulates the significance of early reproduction appears to decrease with increasing survival. Overall, this study supports the theory that survival prospects of males modulate the investments into reproduction via alternative mating tactics early in life. In the case of male Alpine ibex, the results indicate that their life-history strategy targets for long life, slow and prolonged growth and late reproduction.

Prescription patterns of antimicrobials in veterinary practices in Switzerland

OBJECTIVES The objective of this study was to analyse antimicrobial prescriptions by veterinarians and to evaluate the appropriateness of use compared with prudent use guidelines. PRACTICES AND METHODS: Computerized records of prescriptions and treatments from eight mixed veterinary practices were analysed over a period of 2 years. A total of 61 212 antimicrobial treatments were recorded. Treatments were classified according to animal species treated, indication for treatment, route of administration and antimicrobial class used. For each treatment and antimicrobial substance, the prescribed dose was calculated. Dosage, antimicrobial classes and combinations of different classes used for different indications were compared with published recommendations. RESULTS From the total amount of 1590 kg of active antimicrobial substance, sulphonamides (594 kg), tetracyclines (335 kg), and penicillins and cephalosporins (290 kg) were the classes of which the largest quantity was prescribed. Penicillins and cephalosporins were most frequently prescribed (37% of treatments), followed by aminoglycosides (18%), tetracyclines (14%) and sulphonamides (11%). Sixty-one per cent of the amount of antimicrobials prescribed was used for the treatment of groups of animals via feed or water. Antimicrobial classes classified as highest priority for human medicine by an international group of experts were used in 9% of the prescriptions. The dosage corresponded to the manufacturer's recommendation in 45% of the analysed prescriptions. CONCLUSIONS Most prescriptions corresponded well to guidelines on prudent use of antimicrobials. Nevertheless, the large variation of prescriptions among different veterinarians indicates that the usage of critical antimicrobial substances and the amount of antimicrobials used for group medication without a specific indication could be further reduced.

Data quality of animal health records on Swiss dairy farms

High-quality data are essential for veterinary surveillance systems, and their quality can be affected by the source and the method of collection. Data recorded on farms could provide detailed information about the health of a population of animals, but the accuracy of the data recorded by farmers is uncertain. The aims of this study were to evaluate the quality of the data on animal health recorded on 97 Swiss dairy farms, to compare the quality of the data obtained by different recording systems, and to obtain baseline data on the health of the animals on the 97 farms. Data on animal health were collected from the farms for a year. Their quality was evaluated by assessing the completeness and accuracy of the recorded information, and by comparing farmers' and veterinarians' records. The quality of the data provided by the farmers was satisfactory, although electronic recording systems made it easier to trace the animals treated. The farmers tended to record more health-related events than the veterinarians, although this varied with the event considered, and some events were recorded only by the veterinarians. The farmers' attitude towards data collection was positive. Factors such as motivation, feedback, training, and simplicity and standardisation of data collection were important because they influenced the quality of the data.

A Foraging Cost of Migration for a Partially Migratory Cyprinid Fish

Migration has evolved as a strategy to maximise individual fitness in response to seasonally changing ecological and environmental conditions. However, migration can also incur costs, and quantifying these costs can provide important clues to the ultimate ecological forces that underpin migratory behaviour. A key emerging model to explain migration in many systems posits that migration is driven by seasonal changes to a predation/growth potential (p/g) trade-off that a wide range of animals face. In this study we assess a key assumption of this model for a common cyprinid partial migrant, the roach Rutilus rutilus, which migrates from shallow lakes to streams during winter. By sampling fish from stream and lake habitats in the autumn and spring and measuring their stomach fullness and diet composition, we tested if migrating roach pay a cost of reduced foraging when migrating. Resident fish had fuller stomachs containing more high quality prey items than migrant fish. Hence, we document a feeding cost to migration in roach, which adds additional support for the validity of the p/g model of migration in freshwater systems.

Economic efficiency analysis of different strategies to control post-weaning multi-systemic wasting syndrome and porcine circovirus type 2 subclinical infection in 3-weekly batch system farms.

The study assessed the economic efficiency of different strategies for the control of post-weaning multi-systemic wasting syndrome (PMWS) and porcine circovirus type 2 subclinical infection (PCV2SI), which have a major economic impact on the pig farming industry worldwide. The control strategies investigated consisted on the combination of up to 5 different control measures. The control measures considered were: (1) PCV2 vaccination of piglets (vac); (2) ensuring age adjusted diet for growers (diets); (3) reduction of stocking density (stock); (4) improvement of biosecurity measures (bios); and (5) total depopulation and repopulation of the farm for the elimination of other major pathogens (DPRP). A model was developed to simulate 5 years production of a pig farm with a 3-weekly batch system and with 100 sows. A PMWS/PCV2SI disease and economic model, based on PMWS severity scores, was linked to the production model in order to assess disease losses. This PMWS severity scores depends on the combination post-weaning mortality, PMWS morbidity in younger pigs and proportion of PCV2 infected pigs observed on farms. The economic analysis investigated eleven different farm scenarios, depending on the number of risk factors present before the intervention. For each strategy, an investment appraisal assessed the extra costs and benefits of reducing a given PMWS severity score to the average score of a slightly affected farm. The net present value obtained for each strategy was then multiplied by the corresponding probability of success to obtain an expected value. A stochastic simulation was performed to account for uncertainty and variability. For moderately affected farms PCV2 vaccination alone was the most cost-efficient strategy, but for highly affected farms it was either PCV2 vaccination alone or in combination with biosecurity measures, with the marginal profitability between 'vac' and 'vac+bios' being small. Other strategies such as 'diets', 'vac+diets' and 'bios+diets' were frequently identified as the second or third best strategy. The mean expected values of the best strategy for a moderately and a highly affected farm were £14,739 and £57,648 after 5 years, respectively. This is the first study to compare economic efficiency of control strategies for PMWS and PCV2SI. The results demonstrate the economic value of PCV2 vaccination, and highlight that on highly affected farms biosecurity measures are required to achieve optimal profitability. The model developed has potential as a farm-level decision support tool for the control of this economically important syndrome.

Influence of manure scrapers on dairy cows in cubicle housing systems

Manure scrapers are widely used in dairy cow loose-housing systems. In order to evaluate the effects of the scrapers on the cows, we assessed their impact on the animals' cardiac activity, feeding behaviour, and the behavioural reactions of cows confronted with different types of scrapers. In part I of the study, we measured cardiac activity (mean R–R interval and RMSSD, a parameter of heart-rate variability) whilst observing the behaviour of 29 focal cows on three farms during situations with and without active manure scrapers. Lower RMSSD values were observed during scraping events while cows were either lying, standing or walking in the alleyway, standing completely in the lying cubicle, or standing half in the lying cubicle (P=0.03), but only tended to differ while directly confronted with the scraper (P=0.06). This indicates that dairy cows experienced at least some mild stress during manure-scraping events. In part II, the feeding behaviour of 12 cows on each of two farms was recorded by means of a jaw-movement sensor and compared between situations with the manure-scraping event following forage provision either within or outside the main daily feeding period (i.e. within 1 or after 2 h from forage provisioning, respectively). The duration of night-time feeding (P=0.049) and the number of feeding bouts (P=0.036) were higher when a manure-scraping event took place within the main daily feeding period, indicating that the cows' feeding behaviour had been disturbed. In part III, we observed the cows' behaviour on 15 farms during eight manure scraping events per farm, where each of five farms had one of three different scraper types. We assessed the cows' immediate reactions when confronted with the scraper. In addition, we recorded the number of animals present in the alleyways before and after the manure-scraping events. The more cows that were present in the alleyways before the scraping event, the lower the proportion of cows showing direct behavioural reactions both with (P=0.017) and without (P=0.028) scraper contact, and the higher the number of cows that left the alleyways (P<0.001). Scraper type did not influence the proportion of cows showing behavioural reactions. In conclusion, our results show that dairy cows perceive the manure-scraping event negatively in some situations, that feeding behaviour may be disturbed when scrapers are active during the main feeding period, and that cows avoid the scraper during crowded situations.

Morphological characterization of the Azikheli buffalo in Pakistan

This study aims to characterize Azikheli, an undocumented buffalo breed, in its home tract (Khwazakhela, Swat, Pakistan) under traditional farming conditions. For this purpose, 108 buffalo cows and 27 bulls were randomly selected. Mean, standard error, Student's t test and Chi-square test were used for various comparisons. The results show that the majority of animals have a brown coat colour. Cows have significantly higher heart girths, longer horns, longer necks and wider faces at the level of the eyes than bulls, whereas bulls have significantly longer bodies, longer ears, thicker horns, thicker necks and larger hooves than cows. Horns are flat laterally, directed backwards and then slightly upwards without twisting, leading to a sickle to semi-sickle appearance. Owing to its small body size and brown coat colour, the breed is well adapted to mountain slope grazing and thrives well away from swamps. Its adaptation to mountainous ecosystems warrants its in situ conservation.

Characterization and function of murine Thy-1$\sp+$ dendritic epidermal cells

The skin immune system is believed to be a crucial site of contact between immunocompetent cells and invading organisms. A novel T cell component of murine epidermis is the Thy-1$\sp+$ dendritic epidermal cell (Tdec). To assess the immunocompetence of Tdec, the ability of Tdec to induce immune responses was tested. Tdec were unable to induce positive immune responses in three models of immunocompetence. Subsequent studies were designed to test the hypothesis that Tdec are involved in the down-regulation of cell-mediated immunity against cutaneous antigens. Cultured Tdec lines were conjugated in vitro with the hapten, fluorescein isothiocyanate (FITC). The intrafootpad (ifp.) or intravenous (i.v.) injection of FTIC-conjugated Tdec induced immunologic tolerance to subsequent epicutaneous sensitization with FITC. This induction of tolerance was antigen-specific, and injection of unconjugated Tdec had no effect on the contact hypersensitivity response to FITC. Tolerance was not H-2-restricted, since it could be induced in both syngeneic and allogeneic recipients of FITC-conjugated Tdec. No suppressive activity could be detected in lymphoid organs of animals tolerized by the ifp. injection of hapten-conjugated Tdec. In contrast, suppressor T cells were present in the spleens of mice injected i.v. with hapten-conjugated Tdec. These results indicate that Ts cells are not involved in the induction of tolerance by the ifp. injection of hapten-conjugated Tdec. To investigate the mechanism by which the ifp. injection of hapten-conjugated Tdec induced tolerance to contact sensitization, the activity of these cells was measured in vitro. The addition of hapten-conjugated Tdec inhibited the proliferation of Con A-stimulated lymphocytes. In addition, FITC-conjugated Tdec abrogated the proliferation of normal lymphocytes in response to FITC-labeled stimulator cells. These studies suggest that specific T cell-mediated immunity is the target of the inhibitory effect of Tdec in vitro. In summary, these results demonstrate that while Tdec are unable to induce positive immune responses, they can produce a state of specific immunologic tolerance when injected ifp. or i.v. These results also suggest that the induction of immunologic tolerance by hapten-conjugated Tdec may occur through the inactivation or elimination of activated T lymphocytes resulting in down-regulation of cell-mediated immunity against cutaneous antigens. ^

Development of prostate cancer gene therapy

Osseous metastases account for most of the morbidity and mortality associated with prostate cancer, for which there are currently no effective therapies. In the skeletal metastatic environment, neoplastic prostatic epithelial cells interact in a bidirectional stimulatory manner with osteoblastic stromal cells. Similarly, the presence of osteoblastic cells is essential for the survival and maintenance of intraosseous prostate cancer cells. In this thesis, I have developed novel gene therapy strategies for the treatment of androgen-independent human prostate cancers in experimental animal models. First, Ad-CMV-p53, a recombinant adenovirus (Ad) containing p53 tumor suppressor gene driven by the universal cytomegalovirus promoter, was effective in inhibiting prostate cancer cell growth, and direct intratumoral injections of Ad-CMV-p53 resulted in tumor regression. Second, because prostate cancer cells as well as osteoblastic cells produce osteocalcin (OC), OC promoter mediated tissue/tumor specific toxic gene therapy is developed to interrupt stromal-epithelial communications by targeting both cell types. Ad-OC-TK, a recombinant Ad containing the herpes simplex virus thymidine kinase (TK) gene driven by the OC promoter, was generated to inhibit the growth of osteoblastic osteosarcoma with prodrug acyclovir (ACV). Ad-OC-TK/ACV also inhibited the growth of prostate cancer cells and suppressed the growth of subcutaneous and intraosseous prostate tumor. In order to combine treatment modalities to maximize tumor cell-kill with minimized host toxicities, Ad-OC-TK/ACV was applied in combination with low dose methotrexate to eradicate osteoblastic osteosarcoma. In targeting of micrometastatic disease, intravenous Ad-OC-TK/ACV treatment resulted in significant tumor nodule reduction and prolonged the survival of animals harboring osteosarcoma lung metastases without significant host toxicity. Ad-OC-TK is a rational choice for the treatment of prostate cancer skeletal metastasis because OC is uniformly detected in both primary and metastatic human prostate cancer specimens by immunohistochemistry. Ad-OC-TK/ACV inhibits the growth not only of prostate cancer cells but also of their supporting bone stromal cells. Targeting both prostate cancer epithelium and its supporting stroma may be most efficacious for the treatment of prostate cancer osseous metastases. ^

Diffusion tensor imaging of rat spinal cord in vivo

Magnetic resonance imaging, with its exquisite soft tissue contrast, is an ideal modality for investigating spinal cord pathology. While conventional MRI techniques are very sensitive for spinal cord pathology, their specificity is somewhat limited. Diffusion MRI is an advanced technique which is a very sensitive and specific indicator of the integrity of white matter tracts. Diffusion imaging has been shown to detect early ischemic changes in white matter, while conventional imaging demonstrates no change. By acquiring the complete apparent diffusion tensor (ADT), tissue diffusion properties can be expressed in terms of quantitative and rotationally invariant parameters. ^ Systematic study of SCI in vivo requires controlled animal models such as the popular rat model. To date, studies of spinal cord using ADT imaging have been performed exclusively in fixed, excised spinal cords, introducing inevitable artifacts and losing the benefits of MRI's noninvasive nature. In vivo imaging reflects the actual in vivo tissue properties, and allows each animal to be imaged at multiple time points, greatly reducing the number of animals required to achieve statistical significance. Because the spinal cord is very small, the available signal-to-noise ratio (SNR) is very low. Prior spin-echo based ADT studies of rat spinal cord have relied on high magnetic field strengths and long imaging times—on the order of 10 hours—for adequate SNR. Such long imaging times are incompatible with in vivo imaging, and are not relevant for imaging the early phases following SCI. Echo planar imaging (EPI) is one of the fastest imaging methods, and is popular for diffusion imaging. However, EPI further lowers the image SNR, and is very sensitive to small imperfections in the magnetic field, such as those introduced by the bony spine. Additionally, The small field-of-view (FOV) needed for spinal cord imaging requires large imaging gradients which generate EPI artifacts. The addition of diffusion gradients introduces yet further artifacts. ^ This work develops a method for rapid EPI-based in vivo diffusion imaging of rat spinal cord. The method involves improving the SNR using an implantable coil; reducing magnetic field inhomogeneities by means of an autoshim, and correcting EPI artifacts by post-processing. New EPI artifacts due to diffusion gradients described, and post-processing correction techniques are developed. ^ These techniques were used to obtain rotationally invariant diffusion parameters from 9 animals in vivo, and were validated using the gold-standard, but slow, spinecho based diffusion sequence. These are the first reported measurements of the ADT in spinal cord in vivo . ^ Many of the techniques described are equally applicable toward imaging of human spinal cord. We anticipate that these techniques will aid in evaluating and optimizing potential therapies, and will lead to improved patient care. ^

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^