Genomic evidence for the suitability of Göttingen Minipigs with a rare seizure phenotype as a model for human epilepsy.

Epilepsy is a complex genetic disorder that affects about 2% of the global population. Although the frequency and severity of epileptic seizures can be reduced by a range of pharmacological interventions, there are no disease-modifying treatments for epilepsy. The development of new and more effective drugs is hindered by a lack of suitable animal models. Available rodent models may not recapitulate all key aspects of the disease. Spontaneous epileptic convulsions were observed in few Göttingen Minipigs (GMPs), which may provide a valuable alternative animal model for the characterisation of epilepsy-type diseases and for testing new treatments. We have characterised affected GMPs at the genome level and have taken advantage of primary fibroblast cultures to validate the functional impact of fixed genetic variants on the transcriptome level. We found numerous genes connected to calcium metabolism that have not been associated with epilepsy before, such as ADORA2B, CAMK1D, ITPKB, MCOLN2, MYLK, NFATC3, PDGFD, and PHKB. Our results have identified two transcription factor genes, EGR3 and HOXB6, as potential key regulators of CACNA1H, which was previously linked to epilepsy-type disorders in humans. Our findings provide the first set of conclusive results to support the use of affected subsets of GMPs as an alternative and more reliable model system to study human epilepsy. Further neurological and pharmacological validation of the suitability of GMPs as an epilepsy model is therefore warranted.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Quantification of regional myocardial mean intracellular water lifetime: A nonhuman primate study in myocardial stress.

Heart failure with preserved ejection fraction (HFpEF) is typically associated with early metabolic remodeling. Noninvasive imaging biomarkers that reflect these changes will be crucial in determining responses to early drug interventions in these patients. Mean intracellular water lifetime (τi ) has been shown to be partially inversely related to Na, K-ATPase transporter activity and may thus provide insight into the metabolic status in HFpEF patients. Here, we aim to perform regional quantification of τi using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) in the nonhuman primate (NHP) heart and evaluate its region-specific variations under conditions of myocardial stress in the context of perturbed myocardial function. Cardiac stress was induced in seven naïve cynomolgus macaques using a dobutamine stepwise infusion protocol. All animals underwent 3 T cardiac dual-bolus DCE and tagging MRI experiments. The shutter-speed model was employed to quantify regional τi from the DCE-MR images. Additionally, τi values were correlated with myocardial strains. During cardiac stress, there was a significant decrease in global τi (192.9 ± 76.3 ms vs 321.6 ± 70 ms at rest, P < 0.05) in the left ventricle, together with an increase in global peak circumferential strain (-15.4% ± 2.7% vs -10.1% ± 2.9% at rest, P < 0.05). Specifically, slice-level analysis further revealed that a greater significant decrease in mean τi was observed in the apical region (ΔτI = 182.4 ms) compared with the basal (Δτi = 113.2 ms) and midventricular regions (Δτi = 108.4 ms). Regional analysis revealed that there was a greater significant decrease in mean τi in the anterior (Δτi = 243.9 ms) and antero-lateral (Δτi = 177.2 ms) regions. In the inferior and infero-septal regions, although a decrease in τi was observed, it was not significant. Whole heart regional quantification of τi is feasible using DCE-MRI. τi is sensitive to regional changes in metabolic state during cardiac stress, and its value correlates with strain.

Quantitative MRI biomarkers to characterize regional left ventricular perfusion and function in nonhuman primates during dobutamine-induced stress: A reproducibility and reliability study.

PURPOSE: To identify reproducible and reliable noninvasive regional imaging biomarkers of cardiac function and perfusion at rest and under stress in healthy nonhuman primates (NHPs) that may be used in the future for the early characterization of preclinical heart failure models, to evaluate therapy, and for clinical translation. MATERIALS AND METHODS: Seven naive cynomolgus macaques underwent test-retest 3T cardiac MRI tagging and dual-bolus perfusion experiments. Regional cardiac function biomarkers, such as peak circumferential strain (CS), average diastolic strain-rate (DSR), contractile reserve (CR), diastolic reserve, peak torsion, and torsion reserve were quantified. Further, regional myocardial blood flow (MBF), myocardial perfusion reserve (MPR), and myocardial perfusion reserve-to-contractile reserve (MPR/CR) were also derived. Inter- and intraobserver reproducibility and test-retest reliability analyses were conducted using the reliability and generalizability coefficients including correlation coefficient (CC) and intraclass correlation coefficient (ICC). RESULTS: Overall, peak CS, DSR, and MBF are robust biomarkers at both rest and stress with moderate-good inter- and intraobserver reproducibility and test-retest reliability. At rest: intra-/interobserver reproducibility (CC): peak CS (0.81/0.81), DSR (0.81/0.81), MBF (0.72/0.57), peak torsion (0.79/0.79); test-retest reliability: (CC/ICC): peak CS (0.62/0.75), DSR (0.24/0.55), MBF (0.66/0.62), and peak torsion (0.79/0.78). Under stress: intra-/interobserver reproducibility (CC): peak CS (0.61/0.60), DSR (0.50/0.50), MBF (0.63/0.61), MPR (0.43/0.43), and peak torsion (0.38/0.38); test-retest reliability: (CC/ICC): peak CS (0.58/0.58), DSR (0.24/0.43), MBF (0.58/0.58), MPR (0.43/0.38), and peak torsion (0.38/0.38). CONCLUSION: We demonstrated the feasibility of using cardiac MRI to characterize left ventricular functional and perfusion responses to stress in an NHP species, and specific robust biomarkers such as peak CS, DSR, MBF, diastolic reserve, and MPR have been identified for clinical translation and drug research. LEVEL OF EVIDENCE: 1 J. Magn. Reson. Imaging 2017;45:556-569.

First virological and pathological study of Göttingen Minipigs with Dippity Pig Syndrome (DPS).

Dippity Pig Syndrome (DPS) is a well-known but rare complex of clinical signs affecting minipigs, which has not been thoroughly investigated yet. Clinically affected animals show acute appearance of red, exudating lesions across the spine. The lesions are painful, evidenced by arching of the back (dipping), and the onset of clinical signs is generally sudden. In order to understand the pathogenesis, histological and virological investigations were performed in affected and unaffected Göttingen Minipigs (GöMPs). The following DNA viruses were screened for using PCR-based methods: Porcine cytomegalovirus (PCMV), which is a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3), porcine circoviruses (PCV1, PCV2, PCV3, PCV4), porcine parvovirus 1 (PPV1), and Torque Teno sus viruses (TTSuV1, TTSuV2). Screening was also performed for integrated porcine endogenous retroviruses (PERV-A, PERV-B, PERV-C) and recombinant PERV-A/C and their expression as well as for the RNA viruses hepatitis E virus (HEV) and SARS-CoV-2. Eight clinically affected and one unaffected GöMPs were analyzed. Additional unaffected minipigs had been analyzed in the past. The analyzed GöMPs contained PERV-A and PERV-B integrated in the genome, which are present in all pigs and PERV-C, which is present in most, but not all pigs. In one affected GöMPs recombinant PERV-A/C was detected in blood. In this animal a very high expression of PERV mRNA was observed. PCMV/PRV was found in three affected animals, PCV1 was found in three animals with DPS and in the unaffected minipig, and PCV3 was detected in two animals with DPS and in the unaffected minipig. Most importantly, in one animal only PLHV-3 was detected. It was found in the affected and unaffected skin, and in other organs. Unfortunately, PLHV-3 could not be studied in all other affected minipigs. None of the other viruses were detected and using electron microscopy, no virus particles were found in the affected skin. No porcine virus RNA with exception of PERV and astrovirus RNA were detected in the affected skin by next generation sequencing. This data identified some virus infections in GöMPs with DPS and assign a special role to PLHV-3. Since PCMV/PRV, PCV1, PCV3 and PLHV-3 were also found in unaffected animals, a multifactorial cause of DPS is suggested. However, elimination of the viruses from GöMPs may prevent DPS.

The effect of isoflurane anaesthesia and vasectomy on circulating corticosterone and ACTH in BALB/c mice.

The use of blood corticosterone and faecal corticosterone metabolites as biomarkers of post-surgical stress and pain in laboratory animals has increased during the last decade. However, many aspects of their reliability in laboratory mice remain uninvestigated. This study investigated serum corticosterone and adrenocorticotropic hormone (ACTH) in mice subjected to isoflurane anaesthesia and vasectomy, and mice subjected to isoflurane anaesthesia without surgery. Serum levels of corticosterone and ACTH after pre-treatment with dexamethasone were analysed to provide further information about the stress hormone profiles. Vasectomy resulted in an increase in corticosterone for at least four hours after surgery with a peak 30min after the mice regained righting reflex. Mice subjected to isoflurane anaesthesia without surgery had the highest level of serum corticosterone 5min after regained righting reflex and the level returned to baseline levels four hours after the procedure. In vasectomised mice, treated with dexamethasone, high levels of corticosterone remained 30min after the procedure, whereas the anaesthetised mice, treated with dexamethasone, had significantly lower levels of corticosterone compared to anaesthetised mice not treated with dexamethasone. Thus, dexamethasone effectively inhibited the corticosterone response in the anaesthetised-only mice, but not in the mice subjected to surgery. In conclusion, both isoflurane anaesthesia and vasectomy during isoflurane anaesthesia resulted in an increase in serum glucocorticoids, but the negative feedback mechanism of newly operated mice, was altered. This may have consequences for the interpretation of glucocorticoids measurements as a biomarker of post-surgical stress in mice.

Physiological and Welfare Consequences of Transport, Relocation, and Acclimatization of Chimpanzees (Pan troglodytes).

Manipulations of the environments of captive nonhuman primates often have welfare consequences to the animals, including behavioral effects, and for certain manipulations, physiological effects as well. The processes of transporting, relocating, and acclimatizing nonhuman primates across facilities represent manipulations that are likely to have welfare, behavioral, and physiological consequences to the relocated animals. Seventy-two chimpanzees were relocated from the Primate Foundation of Arizona (PFA) in Arizona to the Keeling Center (KCCMR) in Texas. Animals were transported for approximately 21 h in single cages in a USDA-approved, climate-controlled trailer. Chimpanzees were weighed, anesthetized, and blood samples were collected 1) immediately prior to departure from PFA, 2) immediately upon arrival at the KCCMR, and 3) at additional time point(s) between 3 and 12 weeks after arrival at the KCCMR. Chimpanzees were quarantined in familiar pairs or social groups for 60-90 days at the KCCMR. Blood samples were analyzed for hematological and clinical chemistry parameters and compared across time points. In addition, samples from a subset of animals were assayed for cell-mediated immune parameters. Comparisons of the data obtained just prior to transport, to the data obtained immediately upon arrival, revealed numerous statistically significant differences in hematological, clinical chemistry, and immunological parameters. Some of these were indicative of stress, and thus, changes in welfare state, although many remained within the published normal ranges for chimpanzees. Additional analyses showed that many of the clinical chemistry values collected 3 to 12 weeks after arrival at the KCCMR had returned to pre-transport values. In contrast, of the cell-mediated immune parameters that were affected by transport and relocation, few had returned to pre-transport levels 8 weeks after transport, and three of the four hematology variables analyzed had not returned to pre-transport levels 12 weeks after transport. Comparisons of body weights before and immediately after transport revealed that animals lost an average of 2.5 kg during the 21-h transport, a statistically significant reduction that some animals never regained. These results demonstrate that transport and relocation affect a variety of physiological parameters with potential welfare implications and that some of these effects last as long as 3 months. These findings have important implications for the welfare and use of recently transported nonhuman primates, especially chimpanzees, in biomedical research. In order to allow animals to adapt to their new surroundings and to prevent unwanted confounds from influencing experiments, sufficient time must be provided after transport for chimpanzees to acclimatize.

A genetically modified minipig model for Alzheimer's disease with SORL1 haploinsufficiency.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of ß-amyloid (Aß) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Region-specific regulation of 5-HT1A receptor expression by Pet-1-dependent mechanisms in vivo.

Serotonin (5-hydroxytryptamine, 5-HT) neurotransmission is negatively regulated by 5-HT1A autoreceptors on raphe neurons, and is implicated in mood disorders. Pet-1/FEV is an ETS transcription factor expressed exclusively in serotonergic neurons and is essential for serotonergic differentiation, although its regulation of 5-HT receptors has not yet been studied. Here, we show by electrophoretic mobility shift assay that recombinant human Pet-1/FEV binds directly to multiple Pet-1 elements of the human 5-HT1A receptor promoter to enhance its transcriptional activity. In luciferase reporter assays, mutational analysis indicated that while several sites contribute, the Pet-1 site at -1406 bp had the greatest effect on 5-HT1A promoter activity. To address the effect of Pet-1 on 5-HT1A receptor regulation in vivo, we compared the expression of 5-HT1A receptor RNA and protein in Pet-1 null and wild-type littermate mice. In the raphe nuclei of Pet-1-/- mice tryptophan hydroxylase 2 (TPH2) RNA, and 5-HT and TPH immunostaining were greatly reduced, indicating a deficit in 5-HT production. Raphe 5-HT1A RNA and protein levels were also reduced in Pet-1-deficient mice, consistent with an absence of Pet-1-mediated transcriptional enhancement of 5-HT1A autoreceptors in serotonergic neurons. Interestingly, 5-HT1A receptor expression was up-regulated in the hippocampus, but down-regulated in the striatum and cortex. These data indicate that, in addition to transcriptional regulation by Pet-1 in raphe neurons, 5-HT1A receptor expression is regulated indirectly by alterations in 5-HT neurotransmission in a region-specific manner that together may contribute to the aggressive/anxiety phenotype observed in Pet-1 null mice.

GPI-1046 increases presenilin-1 expression and restores NMDA channel activity.

BACKGROUND: Previously we showed that 6-hydroxydopamine lesions of the substantia nigra eliminate corticostriatal LTP and that the neuroimmunolophilin ligand (NIL), GPI-1046, restores LTP. METHODS: We used cDNA microarrays to determine what mRNAs may be over- or under-expressed in response to lesioning and/or GPI-1046 treatment. Patch clamp recordings were performed to investigate changes in NMDA channel function before and after treatments. RESULTS: We found that 51 gene products were differentially expressed. Among these we found that GPI-1046 treatment up-regulated presenilin-1 (PS-1) mRNA abundance. This finding was confirmed using QPCR. PS-1 protein was also shown to be over-expressed in the striatum of lesioned/GPI-1046-treated rats. As PS-1 has been implicated in controlling NMDA-receptor function and LTP is reduced by lesioning we assayed NMDA mediated synaptic activity in striatal brain slices. The lesion-induced reduction of dopaminergic innervation was accompanied by the near complete loss of NDMA receptor-mediated synaptic transmission between the cortex and striatum. GPI-1046 treatment of the lesioned rats restored NMDA-mediated synaptic transmission but not the dopaminergic innervation. Restoration of NDMA channel function was apparently specific as the sodium channel current density was also reduced due to lesioning but GPI-1046 did not reverse this effect. We also found that restoration of NMDA receptor function was also not associated with either an increase in NMDA receptor mRNA or protein expression. CONCLUSION: As it has been previously shown that PS-1 is critical for normal NMDA receptor function, our data suggest that the improvement of excitatory neurotransmission occurs through the GPI-1046-induced up-regulation of PS-1.

Distribution and time course of corticosterone excretion in faeces and urine of female mice with varying systemic concentrations.

Quantification of corticosterone metabolites excreted in faeces and urine is increasingly being used for assessment of preceding corticosterone concentrations in the circulation. This is a promising approach to non-invasive stress assessment in laboratory rodents. It is however unknown whether the proportions of corticosterone metabolites excreted in faeces and urine may differ, depending on the concentration of corticosterone in blood. This uncertainty undermines the applicability of urinary and faecal corticosterone metabolite measurements as biomarkers for stress. Therefore, the terminal distribution and time course of corticosterone excretion, after intravenous injection of varying corticosterone concentrations, was investigated in female mice. Female BALB/c mice excreted 60% of all corticosterone in the urine with an approximate delay of 5h from tail vein administration. The remaining 40% were excreted in faeces, with an approximate delay of 9h from administration. The faecal/urinary excretion ratio, as well as time course of excretion, remained unaltered by administration of various doses of corticosterone covering the entire physiological range of serum corticosterone. Although currently untested for other strains of mice and species of animals, these findings add credence to the utility of faecal and urinary corticosterone as non-invasive biomarkers for physiological stress.

The effect of environmental enrichment on the behavior of captive tufted capuchin monkeys (Cebus apella).

The authors provided different forms of environmental enrichment to six old laboratory male tufted capuchin monkeys (Cebus apella) and studied the behavior of the monkeys during a baseline period and during three enrichment periods. Each observation period lasted 5 d, with an interval of 6 d between periods. During the first enrichment period, the authors provided Buster cubes and wood cylinders with drilled holes filled with gum arabic. During the second enrichment period, monkeys were provided with a deep litter of bark shavings, and during the third enrichment period, they were given Buster cubes, wood cylinders and bark shavings. When provided with enrichment, the monkeys engaged in natural, species-specific activities and began to exhibit behavioral profiles that more closely resembled those of their natural counterparts. This suggests that their psychological well-being had improved and that group housing combined with environmental enrichment can improve the welfare of old laboratory tufted capuchin monkeys that were previously housed individually.

HES1 regulates 5-HT1A receptor gene transcription at a functional polymorphism: essential role in developmental expression.

Mammalian HES1 and HES5 are abundant in developing CNS and inhibit neurogenesis, while HES6 promotes neurogenesis. An early serotonergic differentiation marker, the 5-HT1A receptor, is repressed by HES5 and DEAF1 which recognize the C(-1019), but not G(-1019) allele of a human 5-HT1A promoter polymorphism associated with mood disorders. We tested whether HES1 and HES6 regulate transcriptional activity at this element. HES1 strongly repressed 5-HT1A transcription in neuronal and non-neuronal cells, while HES6 reversed HES1- and HES5-mediated repression. Mutation of a putative HES consensus site blocked HES1 and HES5, but, unlike HES5, HES1 repressed at the G(-1019) allele. To address its role in vivo, the temporal expression of 5-HT1A receptor RNA and protein was examined in HES1-/- mice, and elevated levels in E12.5 hindbrain and midbrain were observed. Thus, HES1 and HES6 oppositely regulate 5-HT1A receptor transcription and HES1 is required for its correct developmental expression.

Wiring and volume transmission in rat amygdala. Implications for fear and anxiety.

The amygdala plays a key role in anxiety. Information from the environment reaches the amygdaloid basolateral nucleus and after its processing is relayed to the amygdaloid central nucleus where a proper anxiogenic response is implemented. Experimental evidence indicates that in this information transfer a GABAergic interface controls the trafficking of impulses between the two nuclei. Recent work indicates that interneuronal communication can take place by classical synaptic transmission (wiring transmission) and by volume transmission in which the neurotransmitter diffuses and flows through the extracellular space from its site of release and binds to extrasynaptic receptors at various distances from the source. Based on evidence from our laboratory the concept is introduced that neurotransmitters in the amygdala can modulate anxiety involving changes in fear learning and memories by effects on receptor mosaics in the fear circuits through wiring and volume transmission modes of communication.

From the Golgi-Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: wiring and volume transmission.

After Golgi-Cajal mapped neural circuits, the discovery and mapping of the central monoamine neurons opened up for a new understanding of interneuronal communication by indicating that another form of communication exists. For instance, it was found that dopamine may be released as a prolactin inhibitory factor from the median eminence, indicating an alternative mode of dopamine communication in the brain. Subsequently, the analysis of the locus coeruleus noradrenaline neurons demonstrated a novel type of lower brainstem neuron that monosynaptically and globally innervated the entire CNS. Furthermore, the ascending raphe serotonin neuron systems were found to globally innervate the forebrain with few synapses, and where deficits in serotonergic function appeared to play a major role in depression. We propose that serotonin reuptake inhibitors may produce antidepressant effects through increasing serotonergic neurotrophism in serotonin nerve cells and their targets by transactivation of receptor tyrosine kinases (RTK), involving direct or indirect receptor/RTK interactions. Early chemical neuroanatomical work on the monoamine neurons, involving primitive nervous systems and analysis of peptide neurons, indicated the existence of alternative modes of communication apart from synaptic transmission. In 1986, Agnati and Fuxe introduced the theory of two main types of intercellular communication in the brain: wiring and volume transmission (WT and VT). Synchronization of phasic activity in the monoamine cell clusters through electrotonic coupling and synaptic transmission (WT) enables optimal VT of monoamines in the target regions. Experimental work suggests an integration of WT and VT signals via receptor-receptor interactions, and a new theory of receptor-connexin interactions in electrical and mixed synapses is introduced. Consequently, a new model of brain function must be built, in which communication includes both WT and VT and receptor-receptor interactions in the integration of signals. This will lead to the unified execution of information handling and trophism for optimal brain function and survival.

Role of the amygdaloid cholecystokinin (CCK)/gastrin-2 receptors and terminal networks in the modulation of anxiety in the rat. Effects of CCK-4 and CCK-8S on anxiety-like behaviour and [3H]GABA release.

The amygdala plays a key role in fear and anxiety. The intercalated islands are clusters of glutamate-responsive GABAergic neurons rich in cholecystokinin (CCK)-2 receptors which control the trafficking of nerve impulses from the cerebral cortex to the central nucleus of amygdala. In this study, the nature of the CCK-glutamate-GABA interactions within the rat rostral amygdala, and their relevance for anxiety, were studied. CCK/gastrin-like immunoreactive nerve terminals were found to be mainly restricted to the paracapsular intercalated islands and the rostrolateral part of the main intercalated island. Behaviourally, the bilateral microinjection of CCK-4 (0.043-4.3 pmol/side) or CCK-8S (4.3 pmol/side) into the rostrolateral amygdala reduced the open-arm exploration in the elevated plus-maze without affecting locomotion. In contrast, neither CCK-4 nor CCK-8S (0.043-4.3 pmol/side) had any effects in the shock-probe burying test as compared with their saline-treated controls. Biochemically, CCK-4 (0.3 and 1.5 microm), unlike CCK-8S, enhanced significantly the K(+)-stimulated release of [(3)H]GABA from amygdala slices. These effects were fully prevented by prior superfusion of the slices with either the selective CCK-2 receptor antagonist CR2945 (3 microm), or 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), 10 microm, a glutamatergic (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist. It is suggested that CCK modulates glutamate-GABA mechanisms by acting on CCK-2 receptors via volume transmission occurring at the level of the basolateral amygdaloid nucleus and/or by synaptic or perisynaptic volume transmission in the region of the rostrolateral main and paracapsular intercalated islands, resulting in subsequent disinhibition of the central amygdaloid nucleus and anxiety or panic-like behaviour.

Rationale and design of DUAL study: Doxycycline to Upgrade response in light chain (AL) amyloidosis (DUAL): A phase 2 pilot study of a two-pronged approach of prolonged doxycycline with plasma cell-directed therapy in the treatment of AL amyloidosis.

Light chain (AL) amyloidosis is a plasma cell neoplasm associated with insoluble fibril deposition from clonal immunoglobulin chains systemically. The disease is associated with high early mortality and morbidity owing to advanced organ deposition as well as lack of proven de-fibrillogenic therapies. Pre-clinical and retrospective clinical data suggests that doxycycline has benefit in AL amyloidosis. The ongoing DUAL study is a single center, open label, phase 2 study in which patients with AL amyloidosis who are undergoing clone-directed therapy for the underlying neoplasm with oral doxycycline given for 1 year to test the hypothesis that prolonged doxycycline use will be safe, feasible, and lead to reduced early mortality in systemic AL amyloidosis and hasten organ amyloid response. Clinical follow up visits will occur at monthly intervals for systemic AL patients and at 3 monthly intervals for localized AL patients. Blood tests will be collected during these time points for hematologic response assessment. Organ testing will be conducted at 3 monthly intervals and radiologic testing will be conducted at 6 monthly intervals. Research blood samples will be collected at baseline, 6 and 12 months. Other correlative studies include matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP) testing and patient-reported outcomes.

Adenosine A2A and dopamine D2 heteromeric receptor complexes and their function.

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A -D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the I3 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of I3 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson's disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.

Detection of beta-endorphin in the cerebrospinal fluid after intrastriatal microinjection into the rat brain.

We have investigated to what extent microinjected beta-endorphin could migrate from the rat brain parenchyma into the CSF compartment. Exogenous rat beta-endorphin (0.1 nmol) was microinjected into the left striatum 1 mm from the lateral ventricle in anesthetized male rats. CSF samples were collected at different time points up to 2 h post-injection from a catheter affixed to the atlanto-occipital membrane of the cisterna magna. Radioimmunoassay and mass spectrometry were performed on the CSF samples, and brain sections were immunostained for beta-endorphin and mu-opioid receptors. The beta-endorphin injected rats showed a marked increase in beta-endorphin immunoreactive (IR) material in the CSF, with a peak at 30-45 min post-injection, and this beta-endorphin-IR material existed mainly as the intact beta-endorphin peptide. The immunohistochemistry results revealed the appearance of distinct beta-endorphin-IR cell bodies in the globus pallidus and the bed nucleus of stria terminalis supracapsular part, regions distant from the injection site, at 2 h post-injection of exogenous beta-endorphin. The beta-endorphin-IR in several of the globus pallidus cell bodies colocalized with the mu-opioid receptor-IR at the cell surface. These findings show that upon delivery of synthetic beta-endorphin, there is a significant intracerebral spread of the injected peptide, reaching regions far from the site of injection via diffusion in the extracellular space and flow in the cerebrospinal fluid. This may be of relevance when interpreting studies based on intracerebral injections of peptides, and advances our knowledge regarding the migration of compounds within the brain.

Anxiolytic effects of intra-amygdaloid injection of the D1 antagonist SCH23390 in the rat.

The intercalated islands are intra-amigdaloid clusters of D1 receptor rich GABAergic neurons, which control impulse traffic between the basolateral complex and the central nucleus of the amygdala. As dopaminergic transmission within the amygdala may play a role in anxiety, the effect of the D1 antagonist SCH23390 microinjected mainly close to the rostral intercalated islands in rats was studied, using the White and Black Box test. SCH23390 reduced anxiety by an increase in the latency of the first entry into the black compartment and by an increase in the total time spent in the white compartment of the White and Black Box test, while there was no significant modification of locomotion. It is suggested that blockade of D1 receptors in the rostral intercalated islands may reduce anxiety through a reduction of GABA-mediated dishinibition of the central amygdaloid nucleus.