The Trace Amine 1 receptor knockout mouse: an animal model with relevance to schizophrenia.

Trace amines have been implicated in a number of neuropsychiatric disorders including depression and schizophrenia. Although long known to modulate neurotransmission indirectly through the release of catecholamines, the identification of the Trace Amine 1 receptor (TA1) offers a mechanism by which trace amines can influence synaptic activity directly. TA1 binds and is activated by trace amines such as beta-phenylethylamine and tyramine. Our pharmacological characterization of mouse TA1 showed that, as in rat and primate, amphetamine is an agonist at this receptor but with surprisingly high potency. Without selective ligands for TA1 that do not also possess catecholamine-releasing properties, however, it has not been possible to study its physiological role in the central nervous system. To that end, a line of mice lacking the TA1 receptor was generated to characterize its contribution to the regulation of behavior. Compared with wild-type littermates, TA1 knockout (KO) mice displayed a deficit in prepulse inhibition. Knockout animals, in which the TA1-agonist influence of amphetamine was absent, showed enhanced sensitivity to the psychomotor-stimulating effect of this drug, which was temporally correlated with significantly larger increases in the release of both dopamine and norepinephrine in the dorsal striatum and associated with a 262% increase in the proportion of striatal high-affinity D2 receptors. TA1 therefore appears to play a modulatory role in catecholaminergic function and represents a potentially novel mechanism for the treatment of neuropsychiatric disorders. Furthermore, the TA1 KO mouse may provide a useful model for the development of treatments for some positive symptoms of schizophrenia.

Efficacy of the MCHR1 antagonist N-[3-(1-{[4-(3,4-difluorophenoxy)phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847) in mouse models of anxiety and depression following acute and chronic administration is independent of hippocampal neurogenesis.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a role in the modulation of food intake and mood. In rodents, the actions of MCH are mediated via the MCHR1 receptor. The goal of this study was to investigate the effects of acute (1 h) and chronic (28 days) p.o. dosing of a novel MCHR1 antagonist, N-[3-(1-{[4-(3,4-difluorophenoxy)-phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847), in three mouse models predictive of antidepressant/anxiolytic-like activity: novelty suppressed feeding (NSF) in 129S6/SvEvTac mice and light/dark paradigm (L/D) and forced swim test (FST) in BALB/cJ mice. A significant increase in the time spent in the light compartment of the L/D box was observed in response to acute and chronic treatment with SNAP 94847. An anxiolytic/antidepressant-like effect was found in the NSF test after acute and chronic treatment, whereas no effect was observed in the FST. Because neurogenesis in the dentate gyrus has been shown to be a requirement for the effects of antidepressants in the NSF test, we investigated whether neurogenesis was required for the effect of SNAP 94847. We showed that chronic treatment with SNAP 94847 stimulated proliferation of progenitors in the dentate gyrus. The efficacy of SNAP 94847 in the NSF test, however, was unaltered in mice in which neurogenesis was suppressed by X-irradiation. These results indicate that SNAP 94847 has a unique anxiolytic-like profile after both acute and chronic administration and that its mechanism of action is distinct from that of selective serotonin reuptake inhibitors and tricyclic antidepressants.

Repeated cocaine administration attenuates group I metabotropic glutamate receptor-mediated glutamate release and behavioral activation: a potential role for Homer.

The present study aimed to characterize a functional role for group I metabotropic glutamate receptors (mGluRs) in the nucleus accumbens and the capacity of repeated cocaine to elicit long-term changes in group I mGluR function. Reverse dialysis of the group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) into the nucleus accumbens resulted in an increase in extracellular glutamate levels that was mediated by the mGluR1 subtype and depended on voltage-dependent Na(+) and Ca(2+) conductance. At 3 weeks after discontinuing 1 week of daily cocaine injections, the capacity of DHPG to induce glutamate release was markedly reduced. Similarly, DHPG induced an mGluR1-dependent increase in locomotor activity after microinjection into the nucleus accumbens that was significantly blunted 3 weeks after repeated cocaine administration. Signaling through group I mGluRs is regulated, in part, by Homer proteins, and it was found that the blunting of group I mGluR-induced glutamate release and motor activity after repeated cocaine was associated with a reduction in Homer1b/c protein that was selective for the medial nucleus accumbens. These data show that repeated cocaine produces an enduring inhibition of the neurochemical and behavioral consequences of stimulating mGluR1 that is accompanied by changes in the mGluR scaffolding apparatus.

CD4+ T cell recognition of a single discordant HLA-A2-transgenic molecule through the indirect antigen presentation pathway induces acute rejection of murine cardiac allografts.

To further define the role of indirect allorecognition, cardiac allografts from HLA-A2-transgenic (HLA-A2+) C57BL/6 mice were heterotopically transplanted into normal C57BL/6, CD4 T cell-knockout (KO) C57BL/6 mice, CD8 T cell-KO C57BL/6 mice, fully MHC-discordant BALB/c mice (allogeneic control), and HLA-A2+ C57BL/6 mice (syngeneic control). HLA-A2+ grafts were acutely rejected when transplanted into BALB/c mice (mean survival time: 10+/-0.8 days), normal C57BL/6 mice (mean survival time: 16.5+/-2.1 days) as well as CD8-KO mice (mean survival time: 12.8+/-1.3 days). Histopathological analysis revealed classical acute cellular rejection with moderate to severe diffuse interstitial CD4+ and CD8+ cellular infiltrates and significant intra-graft deposition of IgG and complement. In contrast, HLA-A2+ grafts were not rejected when transplanted into CD4-KO mice or HLA-A2+ mice. CD8-KO recipients treated with an anti-CD4 monoclonal antibody, but not with an anti-NK monoclonal antibody, failed to reject their allografts with prolonged administration of antibody (30 days). Spleen cells from mice rejecting HLA-A2+ allografts failed to lyse HLA-A2+ target cells indicating a lack of involvement of CD8+ T cells in the rejection process. In contrast, spleen cells from rejecting animals proliferated significantly to both HLA-A2+ cells and to a peptide derived from the HLA-A2 molecule. Development of anti-HLA-A2 antibodies was observed in all animals rejecting HLA-A2+ allografts. These results suggest that indirect allorecognition of donor MHC class I molecules leads to rejection of cardiac allografts and development of alloantibodies in this unique transplant model in which there is a single MHC discordance between donor and recipient.

Improved methods for the isolation and purification of porcine islets.

Recent progress in human islet transplantation demonstrates the feasibility of using purified human islets for treatment of type 1 diabetes mellitus; however, a shortage of human pancreata remains a major obstacle. This report describes methods to isolate porcine islets using a modification of the automated chamber method. The pancreata from 2-year-old sows were trimmed and injected intraductally with Sevac, Sigma, or Liberase PI collagenase. The pancreata was placed in the chamber, shaken, and recirculated at 70 ml/min until an adequate number of islets were liberated. The digest was centrifuged and the pellets pooled with University of Wisconsin Solution + 10% horse serum and incubated at 4 degrees C for 1 h. The islets were purified using a continuous gradient of Hypaque Euroficoll on a refrigerated COBE 2991. The islets were collected in fractions, assessed for purity, sized, and then suspended in Medium 199. Collagenase preparations obtained from Sevac (2919 islet equivalents [IE]/g), Sigma (2543 IE/g), and Liberase PI (2901 IE/g) gave similar results with 94%-95% purity. In summary, we report a successful method for efficient isolation and purification of porcine islets, yielding nearly 3000 IE/gm, with different collagenase products.

Regulation of locomotor activity by metabotropic glutamate receptors in the nucleus accumbens and ventral tegmental area.

Glutamatergic innervation of the ventral tegmental area (VTA) and the nucleus accumbens (NA) regulates locomotor activity. The present study was designed to evaluate the involvement of metabotropic glutamate receptors (mGluRs) in motor activity. Agonists selective for each of the three subgroups of mGluRs were microinjected into the VTA or NA, and motor activity was monitored. The group I agonist (S)-3,5-dihydroxyphenylglycine elicited a dose-dependent elevation in motor activity after microinjection into either the VTA or NA. The effect in the NA was blocked by the mGluR1-specific antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine also elicited a short-duration motor activation after microinjection into either structure. The dose response in the VTA was biphasic, and the coadministration of the group II/III-specific antagonist (RS)-alpha-methyl-4-phosphonophenylglycine partially blocked motor activation in both the NA and VTA. Although the group III agonist L-(+)-2-amino-4-phosphonobutyric acid produced a relatively modest behavioral stimulation after microinjection into the NA, it was without effect in the VTA. These data indicate a role for mGluR subgroups in the regulation of motor activity in the VTA and NA.

Repeated cocaine alters glutamate receptor subunit levels in the nucleus accumbens and ventral tegmental area of rats that develop behavioral sensitization.

Increased glutamate transmission in the nucleus accumbens and ventral tegmental area has been proposed as a mechanism underlying sensitized behavioral responses to repeated cocaine administration. GluR1, GluR2/3, and NMDAR1 subunits of glutamate receptors were quantified from immunoblots in these brain nuclei in rats at 24 h and 3 weeks after discontinuing 1 week of daily cocaine injections. Motor behavior was monitored after the first and last injections of daily cocaine, and those rats that showed >20% increase in motor activity after the last compared with the first injection were considered to have developed behavioral sensitization. The subjects that developed behavioral sensitization showed a significant increase in GluR1 levels in the nucleus accumbens at 3 weeks but not at 24 h of withdrawal. Conversely, sensitized animals showed a significant increase in NMDAR1 and GluR1 levels in the ventral tegmental area at 1 day but not at 3 weeks of withdrawal. None of these increases occurred in the rats exposed to daily cocaine that did not develop behavioral sensitization (<20% increase in motor activity), and no changes were measured in the level of GluR2/3 in any treatment group. The functional importance of the increases in glutamate receptor subunit levels is suggested by the fact that the changes were present only in rats that developed behavioral sensitization to repeated cocaine administration.

Improved method for the isolation and purification of human islets of langerhans using Liberase enzyme blend.

To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of 1Hypaque EuroFicoll. Isolations in Group I (Sevac) had an average yield of 138,602 +/- 128,364 islet equivalents (IE) (2083 +/- 1679 IE/g) with a purity of 85 +/- 11%. Group II (Liberase) showed an average yield of 389,586 +/- 191,161 IE (5,958 +/- 3,083 IE/g) with a purity of 90 +/- 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.

Specific changes in food intake elicited by blockade or activation of glutamate receptors in the nucleus accumbens shell.

Blockade of non-N-methyl-D-aspartic acid (NMDA) ionotropic glutamate receptors in the nucleus accumbens shell (AcbSh) with 6,7-dinitroquinoxaline-2,3-dione (DNQX) elicits intense feeding in satiated rats. In order to determine whether or not this feeding is part of a general behavioral activation, we observed the effect of intra-AcbSh DNQX injections on intake of solid food, liquid food, and water, and on gnawing behavior. In addition, we investigated the possibility that activation of a subset of these receptors with (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) could suppress feeding. DNQX significantly increased intake of solid and liquid food, but did not significantly affect water intake or gnawing behavior. Furthermore, injections of AMPA into the AcbSh suppressed deprivation-induced feeding and intake of a palatable 5% sucrose solution without affecting water intake in water-deprived rats. Taken together, these data suggest that DNQX is acting on a system specifically involved with the regulation of food intake.

Feeding induced by blockade of AMPA and kainate receptors within the ventral striatum: a microinfusion mapping study.

The corticostriatal pathway is believed to utilize the excitatory amino acid glutamate as its transmitter, and the striatum contains high levels of all glutamate receptor subtypes. It has recently been demonstrated that blockade of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptors in the medial part of the accumbens, corresponding to the medial shell subregion, results in a pronounced feeding response. In order to more precisely localize this response, a microinfusion mapping study was conducted. Bilateral microinfusions of 6,7-dinitroquinoxaline-2,3-dione (DNQX, 0, 50, 250, 750 ng/0.5 microl), an antagonist that blocks AMPA and kainate receptors, were carried out in eight striatal subregions in different groups of animals. In non-deprived rats, food intake (normal chow), feeding duration, and several other behavioral measures were assessed during a 30 min test session. DNQX significantly and potently enhanced food intake when injected into the accumbens shell, but not into any other region examined, including accumbens core, anterior dorsal, posterior dorsal, ventromedial, dorsomedial, and ventrolateral striatum. The most sensitive site within the accumbens was found to be the posterior aspects of the shell, in which the lowest dose (50 ng DNQX) augmented feeding. These results suggest that a circumscribed region within the nucleus accumbens has a unique role in the control of feeding. It is postulated that removal of tonic excitatory inputs to this region with DNQX results in disinhibition of brain circuits critical for ingestive behavior.

Differential behavioral responses to dopaminergic stimulation of nucleus accumbens subregions in the rat.

The following experiments investigated the behavioral response to local microinfusion of dopamine (DA) and selective DA agonists into the core and shell subregions of the nucleus accumbens. Rats were implanted with chronic indwelling cannulae aimed at these subregions. Two experiments were conducted. In experiment 1, the response to DA (0, 2, 5, 10 microg/0.5 microl/side), the D-1 agonist SKF-82598 (0, 0.1, 1.0 microg), the D-2/3 agonist quinpirole (0, 1, 5, 15 microg) and the D-3 preferring agonist pramipexole (0.1, 1.0, 10.0 microg) was examined in photocell activity cages. Locomotor (horizontal) and rearing (vertical) activities were measured. DA and SKF-82958 induced relatively greater increases in activity following stimulation of the shell as compared with the core. Quinpirole induced a dose-dependent suppression of activity after infusion into both sites, although the core was more sensitive to the suppressive effect than the shell. Pramipexole induced time-dependent, biphasic effects that were small in magnitude and did not differentiate between site. In experiment 2, an observation procedure was used to record behaviors (locomotion, rearing, feeding, drinking). Dopamine (0, 2, 10 microg) elicited greater increases in rearing and feeding behavior in the shell than in the core. SKF-82958 (0, 0.75 microg) enhanced locomotion and rearing to a similar extent in both subregions in this test, whereas a mixture of a low dose (0.25 microg) of the D-1 and D-2 agonists selectively induced behavioral activation in the shell. In contrast to the results in the activity cage test, quinpirole (0, 1, 5 microg) increased motor activity at the lower dose when infused into the shell but not into the core. No alterations in feeding were observed following infusion of selective agonists, and no changes in drinking were found with any of the treatments. In summary, the shell appears to be relatively more sensitive to the motor activating effects of DA agonists than the core. Moreover, circuits associated with shell may be preferentially involved in feeding.

Investigation of the effects of opiate antagonists infused into the nucleus accumbens on feeding and sucrose drinking in rats.

Previous work has shown that opiate agonist infusion into the nucleus accumbens, a region implicated in reinforcement, stimulates food intake. In the present study, the effects of opiate antagonist infusion into this region were examined in two behavioral paradigms. In the feeding test, food-deprived animals were tested for intake of laboratory chow. In the sucrose intake test, sated animals familiar with a 20% sucrose solution were tested. Before these tests, the following drugs were bilaterally infused into the accumbens: naloxone (0, 1, 10 and 30 micrograms; equivalent to 2.8, 28 and 83 nmol, respectively), naltrexone (0, 0.2, 2 and 20 micrograms; 0.55, 5.5 and 55 nmol, respectively), beta-funaltrexamine (0 and 15 micrograms; 31 nmol), naloxonazine (0 and 10 micrograms; 15 nmol), naltrindole (trial 1: 0, 1, 10 and 20 micrograms; 2.2, 22 and 44 nmol, respectively; trial 2: 0, 0.1 and 0.5 micrograms; 0.22 and 1.1 nmol, respectively) and nor-binaltorphimine (0, 0.1, 1 and 10 micrograms; 0.14, 1.4 and 14 nmol, respectively). Naloxone and naltrexone both significantly reduced sucrose drinking and did not affect feeding. Naloxone infused into the dorsolateral striatum, as a control, had no effect on sucrose drinking. Accumbens infusion of the mu antagonist beta-funaltrexamine reduced both sucrose drinking and feeding. The mu 1 antagonist naloxonazine did not influence intake behaviors, with the exception of a decrease in duration of chow feeding. In contrast, the delta antagonist naltrindole markedly potentiated both sucrose drinking and duration of chow feeding. In a replication of this effect, systemic naltrexone given concurrently blocked the enhancement. The kappa antagonist nor-binaltorphimine did not influence any parameters of ingestive behavior. Although some treatments also decreased motor activities, the overall profile of behavior suggested specific effects on ingestive behavior. The putative contributions of mu and delta receptors within the nucleus accumbens to modulation of food reward are discussed.

Prolonged survival of discordant porcine islet xenografts.

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets.

Glutamate receptors in the nucleus accumbens shell control feeding behavior via the lateral hypothalamus.

The nucleus accumbens in a brain region considered to be important in the regulation of appetitive behavior and reinforcement. The accumbens receives afferent input from corticolimbic and thalamic structures, which is primarily coded by excitatory amino acids (EAAs). The present studies investigated the role of EAA input to the nucleus accumbens in feeding behavior in rats, in two recently characterized subregions of the accumbens, the "core" and "shell". In the first series of experiments, it was shown that blockade of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptors in the medial part of the accumbens, corresponding to the medial shell subregion, resulted in a pronounced feeding response. Bilateral microinfusion of 6,7-dinitroquinoxaline-2,3-dione (DNQX, 0.25-0.75 micrograms/0.5 microliters), 6-cyano-7-nitroquinoxaline (CNQX, 0.75-1.5 micrograms), and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-(F) quinoxaline (NBQX, 0.2-1.0 micrograms) markedly stimulated food intake immediately following infusion, in a dose-dependent manner. Infusion of DNQX into the central accumbens region, corresponding to the core, did not elicit feeding. Infusion of the NMDA antagonists 2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 (dizocilpine maleate) did not elicit feeding in either region. The feeding response to DNQX was blocked by local coinfusion of AMPA. Systemic pretreatment with naltrexone (5 mg/kg) had no effect on the DNQX-feeding response; however, prior systemic administration of both D-1 and D-2 antagonists reduced the response by half, suggesting a modulatory role for dopamine in the response. Moreover, the feeding response was completely inhibited by concurrent infusion of the GABAA agonist muscimol (10, 25 ng) into the lateral hypothalamus, a major projection area of the accumbens shell. These findings demonstrate a selective role for non-NMDA receptors in the nucleus accumbens shell in ingestive behavior, and suggest an important functional link between two major brain regions involved in reward, the nucleus accumbens and lateral hypothalamus.

Protection of encapsulated human islets implanted without immunosuppression in patients with type I or type II diabetes and in nondiabetic control subjects.

Human islets were macroencapsulated in permselective hollow fiber membrane devices and successfully allotransplanted subcutaneously with > 90% viability after 2 weeks in situ. Recipients were patients with type I or type II diabetes and normal control subjects; none was immunosuppressed. Between 150 and 200 islet equivalents were implanted in each of the nine patients. No adverse patient complications were observed. Biocompatibility of devices was excellent. Insulin-positive beta-cells were confirmed in encapsulated islets recovered from the implanted devices in all patient populations including the type I diabetic patients. Glucose-stimulated insulin release could be demonstrated in vitro from recovered islets. These data demonstrate that macroencapsulated human islets can survive at the subcutaneous site and that permselective membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune component of type I diabetes.