Effects of whole-brain radiation therapy on the blood-brain barrier in immunocompetent and immunocompromised mouse models.

BACKGROUND: Approximately 20% of all cancer patients will develop brain metastases in their lifespan. The standard of care for patients with multiple brain metastases is whole-brain radiation therapy, which disrupts the blood-brain barrier. Previous studies have shown inflammatory mediators play a role in the radiation-mediated increase in permeability. Our goal was to determine if differential permeability post-radiation occurs between immunocompetent and immunocompromised mice. METHODS: We utilized a commissioned preclinical irradiator to irradiate brains of C57Bl/6J wild-type and athymic nude mice. Acute (3-24 h) effects on blood-brain barrier integrity were evaluated with our in-situ brain perfusion technique and quantitative fluorescent and phosphorescent microscopy. The presence of inflammatory mediators in the brain and serum was determined with a proinflammatory cytokine panel. RESULTS: Blood-brain barrier integrity and efflux transporter activity were altered in the immunocompetent mice 12 h following irradiation without similar observations in the immunocompromised mice. We observed increased TNF-α concentrations in the serum of wild-type mice immediately post-radiation and nude mice 12 h post-radiation. The brain concentration of CXCL1 was also increased in both mouse strains at the 12-h time point. CONCLUSIONS: The immune response plays a role in the magnitude of blood-brain barrier disruption following irradiation in a time- and size-dependent manner.

Glutamate Afferents From the Medial Prefrontal Cortex Mediate Nucleus Accumbens Activation by Female Sexual Behavior.

Low levels of desire and arousal are the primary sexual dysfunctions in women, necessitating neurobiological studies of sexual motivation in female animal models. As the mesocorticolimbic system is a primary neural circuit underlying sexual motivation, the goal of this study was to test the hypothesis that medial prefrontal cortex (mPFC) glutamate mediates sexual behavior activation of the nucleus accumbens. Glutamatergic neurons in the mPFC were activated by sex behavior, and these sex-activated cells shown to project to the nucleus accumbens. During sexual interactions with the male, glutamate transients recorded in the nucleus accumbens of female hamsters were specifically associated with the receipt of intromissions from the male. Further, inhibition of the mPFC during sex significantly decreased nucleus accumbens activation. Glutamatergic medial prefrontal cortical input to the nucleus accumbens mediates the activity in the nucleus accumbens during female sexual behavior. These results offer novel insights into the neurobiology of the motivational control of female sexual behavior and provide attractive avenues for pursuing target-specific and clinically-relevant therapies for sexual dysfunction in women.

Risk factors for acquisition of multidrug-resistant Escherichia coli and development of community-acquired urinary tract infections.

We examined risk factors associated with the intestinal acquisition of antimicrobial-resistant extraintestinal pathogenic Escherichia coli (ExPEC) and development of community-acquired urinary tract infection (UTI) in a case-control study of young women across Canada. A total of 399 women were recruited; 164 women had a UTI caused by E. coli resistant to ⩾1 antimicrobial classes and 98 had a UTI caused by E. coli resistant to ⩾3 antimicrobial classes. After adjustment for age, student health service (region of Canada) and either prior antibiotic use or UTI history, consumption of processed or ground chicken, cooked or raw shellfish, street foods and any organic fruit; as well as, contact with chickens, dogs and pet treats; and travel to Asia, were associated with an increased risk of UTI caused by antimicrobial resistant E. coli. A decreased risk of antimicrobial resistant UTI was associated with consumption of apples, nectarines, peppers, fresh herbs, peanuts and cooked beef. Drug-resistant UTI linked to foodborne and environmental exposures may be a significant public health concern and understanding the risk factors for intestinal acquisition of existing or newly emerging lineages of drug-resistant ExPEC is important for epidemiology, antimicrobial stewardship and prevention efforts.

Rescue of impaired sociability and anxiety-like behavior in adult cacna1c-deficient mice by pharmacologically targeting eIF2α.

CACNA1C, encoding the Cav1.2 subunit of L-type Ca2+ channels, has emerged as one of the most prominent and highly replicable susceptibility genes for several neuropsychiatric disorders. Cav1.2 channels play a crucial role in calcium-mediated processes involved in brain development and neuronal function. Within the CACNA1C gene, disease-associated single-nucleotide polymorphisms have been associated with impaired social and cognitive processing and altered prefrontal cortical (PFC) structure and activity. These findings suggest that aberrant Cav1.2 signaling may contribute to neuropsychiatric-related disease symptoms via impaired PFC function. Here, we show that mice harboring loss of cacna1c in excitatory glutamatergic neurons of the forebrain (fbKO) that we have previously reported to exhibit anxiety-like behavior, displayed a social behavioral deficit and impaired learning and memory. Furthermore, focal knockdown of cacna1c in the adult PFC recapitulated the social deficit and elevated anxiety-like behavior, but not the deficits in learning and memory. Electrophysiological and molecular studies in the PFC of cacna1c fbKO mice revealed higher E/I ratio in layer 5 pyramidal neurons and lower general protein synthesis. This was concurrent with reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α, an inhibitor of mRNA translation. Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC. Thus this study identifies a novel Cav1.2 mechanism in neuropsychiatric-related endophenotypes and a potential future therapeutic target to explore.

Serotonin in the dorsal periaqueductal gray inhibits panic-like defensive behaviors in rats exposed to acute hypoxia.

It has been proposed that spontaneous panic attacks are the outcome of the misfiring of an evolved suffocation alarm system. Evidence gathered in the last years is suggestive that the dorsal periaqueductal gray (dPAG) in the midbrain harbors a hypoxia-sensitive suffocation alarm system. We here investigated whether facilitation of 5-HT-mediated neurotransmission within the dPAG changes panic-like defensive reactions expressed by male Wistar rats submitted to a hypoxia challenge (7% O2), as observed in other animal models of panic. Intra-dPAG injection of 5-HT (20 nmol), (±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT) (8 nmol), a 5-HT1A receptor agonist, or (±)-2,5-dimethoxy-4-iodo amphetamine hydrochloride (DOI) (16 nmol), a preferential 5-HT2A agonist, reduced the number of upward jumps directed to the border of the experimental chamber during hypoxia, interpreted as escape attempts, without affecting the rats' locomotion. These effects were similar to those caused by chronic, but not acute, intraperitoneal administration of the antidepressant fluoxetine (5-15 mg/kg), or acute systemic administration of the benzodiazepine receptor agonist alprazolam (1-4 mg/kg), both drugs clinically used in the treatment of panic disorder. Our findings strengthen the view that the dPAG is a key encephalic area involved in the defensive behaviors triggered by activation of the suffocation alarm system. They also support the use of hypoxia-evoked escape as a model of respiratory-type panic attacks.

Sex differences in NMDA GluN1 plasticity in rostral ventrolateral medulla neurons containing corticotropin-releasing factor type 1 receptor following slow-pressor angiotensin II hypertension.

There are profound, yet incompletely understood, sex differences in the neurogenic regulation of blood pressure. Both corticotropin signaling and glutamate receptor plasticity, which differ between males and females, are known to play important roles in the neural regulation of blood pressure. However, the relationship between hypertension and glutamate plasticity in corticotropin-releasing factor (CRF)-receptive neurons in brain cardiovascular regulatory areas, including the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN), is not understood. In the present study, we used dual-label immuno-electron microscopy to analyze sex differences in slow-pressor angiotensin II (AngII) hypertension with respect to the subcellular distribution of the obligatory NMDA glutamate receptor subunit 1 (GluN1) subunit of the N-methyl-D-aspartate receptor (NMDAR) in the RVLM and PVN. Studies were conducted in mice expressing the enhanced green fluorescence protein (EGFP) under the control of the CRF type 1 receptor (CRF1) promoter (i.e., CRF1-EGFP reporter mice). By light microscopy, GluN1-immunoreactivity (ir) was found in CRF1-EGFP neurons of the RVLM and PVN. Moreover, in both regions tyrosine hydroxylase (TH) was found in CRF1-EGFP neurons. In response to AngII, male mice showed an elevation in blood pressure that was associated with an increase in the proportion of GluN1 on presumably functional areas of the plasma membrane (PM) in CRF1-EGFP dendritic profiles in the RVLM. In female mice, AngII was neither associated with an increase in blood pressure nor an increase in PM GluN1 in the RVLM. Unlike the RVLM, AngII-mediated hypertension had no effect on GluN1 localization in CRF1-EGFP dendrites in the PVN of either male or female mice. These studies provide an anatomical mechanism for sex-differences in the convergent modulation of RVLM catecholaminergic neurons by CRF and glutamate. Moreover, these results suggest that sexual dimorphism in AngII-induced hypertension is reflected by NMDA receptor trafficking in presumptive sympathoexcitatory neurons in the RVLM.

Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy.

Astrocytes are an attractive cell target for gene therapy, but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino-acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks postinjection. ADK expression in miR-ADK vector-injected rats was reduced by 94-96% and was associated with an ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared with miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest that alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.

Central hydrogen sulphide mediates ventilatory responses to hypercapnia in adult conscious rats.

AIM: Hydrogen sulphide (H2S) is endogenously produced and plays an important role as a modulator of neuronal functions; however, its modulatory role in the central CO2 chemoreception is unknown. The aim of the present study was to assess the role of endogenously produced H2S in the ventilatory response to hypercapnia in adult conscious rats. METHODS: Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) inhibitors (aminooxyacetate: AOA and propargylglycine: PAG respectively) and a H2S donor (sodium sulphide: Na2S) were microinjected into the fourth ventricle (4V). Ventilation (V̇(E)), oxygen consumption (V̇O2) and body temperature were recorded before (room air) and during a 30-min CO2 exposure (hypercapnia, 7% CO2). Endogenous H2S levels were measured in the nucleus tractus solitarius (NTS). RESULTS: Microinjection of Na2S (H2S donor), AOA (CBS inhibitor) or PAG (CSE inhibitor) did not affect baseline of the measured variables compared to control group (vehicle). In all experimental groups, hypercapnia elicited an increase in V̇(E). However, AOA microinjection, but not PAG, attenuated the ventilatory response to hypercapnia (P < 0.05), whereas Na2S elicited a slight, not significant, enhancement. Moreover, endogenous H2S levels were found higher in the NTS after hypercapnia (P < 0.05) compared to room air (normoxia) condition. CONCLUSION: There are a few reports on the role of gaseous transmitters in the control of breathing. Importantly, the present data suggest that endogenous H2S via the CBS-H2S pathway mediates the ventilatory response to hypercapnia playing an excitatory role.

Endogenous preoptic hydrogen sulphide attenuates hypoxia-induced hyperventilation.

AIM: We hypothesized that hydrogen sulphide (H2 S), acting specifically in the anteroventral preoptic region (AVPO - an important integrating site of thermal and cardiorespiratory responses to hypoxia in which H2 S synthesis has been shown to be increased under hypoxic conditions), modulates the hypoxic ventilatory response. METHODS: To test this hypothesis, we measured pulmonary ventilation (V˙E) and deep body temperature of rats before and after intracerebroventricular (icv) or intra-AVPO microinjection of aminooxyacetate (AOA; CBS inhibitor) or Na2 S (H2 S donor) followed by 60 min of hypoxia exposure (7% O2 ). Furthermore, we assessed the AVPO levels of H2 S of rats exposed to hypoxia. Control rats were kept under normoxia. RESULTS: Microinjection of vehicle, AOA or Na2 S did not change V˙E under normoxic conditions. Hypoxia caused an increase in ventilation, which was potentiated by microinjection of AOA because of a further augmented tidal volume. Conversely, treatment with Na2 S significantly attenuated this response. The in vivo H2 S data indicated that during hypoxia the lower the deep body temperature the smaller the degree of hyperventilation. Under hypoxia, H2 S production was found to be increased in the AVPO, indicating that its production is responsive to hypoxia. The CBS inhibitor attenuated the hypoxia-induced increase in the H2 S synthesis, suggesting an endogenous synthesis of the gas. CONCLUSION: These data provide solid evidence that AVPO H2 S production is stimulated by hypoxia, and this gaseous messenger exerts an inhibitory modulation of the hypoxic ventilatory response. It is probable that the H2 S modulation of hypoxia-induced hyperventilation is at least in part in proportion to metabolism.

Behavioural and molecular consequences of chronic cannabinoid treatment in Huntington's disease transgenic mice.

Early loss of CB1 receptors is a hallmark of human Huntington's disease. Data from rodent studies suggest that preservation and activation of CB1 receptors may be protective against disease progression. R6/1 transgenic mice are considered to be a model of early pathogenic changes in Huntington's disease. We have shown previously that levels of CB1 in R6/1 mice prior to the onset of motor symptoms (12 weeks of age) remain high enough to justify commencement of cannabinoid drug treatment. Eight weeks of daily treatment with the cannabinoid agonists HU210 (0.01 mg/kg) and Delta(9)-tetrahydrocannabinol (THC, 10.00 mg/kg), or the inhibitor of endocannabinoid metabolism URB597 (0.30 mg/kg), did not alter the progressive deterioration of performance observed in motor behavioural testing. HU210-treated R6/1 mice experienced a significant increase in seizure events suggesting that this therapy may lower the seizure threshold and cautioning against highly efficacious agonists as potential therapy in this disease. Molecular characterisation of brains at the end of the study showed that there were no significant effects of HU210 or THC treatment on the ligand binding of cannabinoid CB1, dopamine D1, D2, serotonin 5HT2A or GABA(A) receptors, nor CB1 or fatty acid amide hydrolase (FAAH) mRNA expression in R6/1 mice. Intriguingly, a significant increase in the number of ubiquitinated aggregates was observed in the striatum with HU210 treatment, indicating an influence of CB1 on the disease process. Chronic URB597 treatment preserved CB1 receptors in the R6/1 striatum, suggesting that the manipulation of endocannabinoid levels warrants further exploration.

Neuroprotective potential of CB1 receptor agonists in an in vitro model of Huntington's disease.

BACKGROUND AND PURPOSE: The therapeutic potential of cannabinoids in Huntington's disease (HD) has been investigated by several groups with complex and sometimes contrasting results. We sought to examine key points of intersection between cannabinoid receptor 1 (CB(1)) signalling, survival and the formation of mutant huntingtin aggregates in HD. EXPERIMENTAL APPROACH: Using a simplified pheochromocytoma (PC12) cell model of HD expressing exon 1 of wild-type or mutant huntingtin, we assayed cell death and aggregate formation using high-throughput cytotoxicity and image-based assays respectively. KEY RESULTS: CB(1) activation by HU210 conferred a small but significant level of protection against mutant huntingtin-induced cell death. Pertussis toxin uncoupled HU210 from the inhibition of cAMP, preventing rescue of cell death. Phosphorylation of extracellular signal-regulated kinase (ERK) was also critical to CB(1)-mediated rescue. Conversely, treatments that elevated cAMP exacerbated mutant huntingtin-induced cell death. Despite opposing effects on HD cell survival, both HU210 and compounds that elevated cAMP increased the formation of mutant huntingtin aggregates. The increase in aggregation by HU210 was insensitive to Pertussis toxin and UO126, suggesting a G-protein alpha subtype s (G(s))-linked mechanism. CONCLUSIONS AND IMPLICATIONS: We suggest that the CB(1) receptor, through G-protein alpha subtype i/o (G(i/o))-linked, ERK-dependent signal transduction, is a therapeutic target in HD. However the protective potential of CB(1) may be limited by promiscuous coupling to G(s), the stimulation of cAMP formation and increased aggregate formation. This may underpin the poor therapeutic efficacy of cannabinoids in more complex model systems and suggest that therapies that are selective for the G(i/o), ERK pathway may be of most benefit in HD.

Detailed characterisation of CB2 receptor protein expression in peripheral blood immune cells from healthy human volunteers using flow cytometry.

It is commonly accepted from gene expression studies that the CB2 receptor is expressed by most cell types of the rodent and human immune system. However, the exact identity of cells expressing CB2 receptor protein in human blood or the abundance of receptors expressed by each immune subset is not well characterised. We conducted a detailed analysis of CB2 protein levels expressed by blood-derived immune cells from healthy human donors. Flow-cytometry was conducted using 4 commercially available anti-CB2 polyclonal antibodies in conjunction with a selection of immune cell specific markers. Across multiple healthy subjects we observed that NK cells, B-lymphocytes and monocytes expressed a higher level of CB2 receptor than CD4+ or CD8+ T-lymphocytes. Neutrophils also expressed a low level of CB2 receptor. NK cells had the greatest variation in CB2 expression levels, whereas for each of the other cell types CB2 levels were relatively similar between subjects. In contrast to other methods, the high sensitivity of flow-cytometry revealed that CB2 receptors are present on resting T-lymphocytes at low abundance in some healthy subjects. These data provide the first detailed analysis of CB2 protein levels in blood leukocyte subsets from healthy donors and identifies the cell types which could be targeted with CB-mimetic drugs in humans.

First Report of Brown Ring Patch on Poa annua Caused by Waitea circinata var. circinata in West Virginia.

In mid-November 2009, thin, yellow, and irregular-shaped scalloped rings 10 to 25 cm in diameter were observed on 5 to 10% of a golf course putting green in Charles Town, WV. The 20-year-old USGA-specification sand-based green was mowed at 3.1-mm height and consisted of 60% annual bluegrass (Poa annua L.) and 40% creeping bentgrass (Agrostis stoloniferous L. 'Putter'). Minimum and maximum daily air temperature ranged from 2 to 22°C, respectively, with 38 mm of rainfall during the appearance of rings symptoms. Only affected annual bluegrass plants exhibited a peculiar yellow chlorosis of the upper and lower leaves. A single fungal isolate was obtained from active mycelium found within symptomatic annual bluegrass leaves and grown on potato dextrose agar (PDA) amended with chloramphenicol (0.1 g/liter). Fungal colony morphology (i.e., light yellow with irregular-shaped 2- to 4-mm-diameter sclerotia first appearing off-white but progressing to brown by 21 to 28 days in culture) and sequencing of the internal transcribed spacer (ITS) 5.8S rDNA region with primers ITS1 and ITS4 confirmed the isolate as Waitea circinata var. circinata (Warcup & Talbot) with ≥99% sequence identity with GenBank Accession No. FJ755889 (1,2,4). To confirm pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 4-day-old culture grown on PDA and placed in contact with the lower leaves of 12-week-old annual bluegrass (0.001 g of surface-sterilized seed per cm2) grown in 5- × 5-cm plastic pots of autoclaved 85% sand and 15% potting soil. Six pots were inoculated with the isolate and six pots were inoculated with an isolate-free agar plug and then placed in a moist chamber at 28°C. Leaf chlorosis and aerial mycelium was observed in all six inoculated pots 8 to 10 days after inoculation, and symptoms were similar to those expressed in the field. All noninoculated plants remained healthy and asymptomatic. W. circinata var. circinata was reisolated from symptomatic leaves and again confirmed by colony traits and sequencing of the ITS-5.8S rDNA region and submitted as GenBank Accession No. HM807582. To our knowledge, this is the first report of brown ring patch in West Virginia and could be economically important because of intensive fungicide practices used to maintain high-quality putting greens on golf courses (3). References: (1) C. Chen et al. Plant Dis. 91:1687, 2007. (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) J. Kaminski and F. Wong. Golf Course Manage. 75:98, 2007. (4) T. Toda et al. Plant Dis. 89:536, 2005.

Ultrastructural relationship between N-methyl-D-aspartate-NR1 receptor subunit and mu-opioid receptor in the mouse central nucleus of the amygdala.

The central nucleus of the amygdala (CeA) is an important neuroanatomical substrate of emotional processes that are critically involved in addictive behaviors. Glutamate and opioid systems in the CeA play significant roles in neural plasticity and addictive processes, however the cellular sites of interaction between agonists of N-methyl-d-aspartate (NMDA) and mu-opioid receptors (muOR) in the CeA are unknown. Dual labeling immunocytochemistry was used to determine the ultrastructural relationship between the essential NMDA-NR1 receptor subunit and muOR in the CeA. It was found that over 80% of NR1-labeled profiles were dendrites while less than 10% were axons. In the case of muOR-labeled profiles, approximately 60% were dendritic, and over 35% were axons. Despite their somewhat distinctive patterns of cellular location, numerous dual-labeled profiles were observed. Approximately 80% of these were dendritic, and less than 10% were axonal. Moreover, many dual-labeled dendritic profiles were contacted by axon terminals receiving asymmetric-type synapses indicative of excitatory signaling. These results indicate that NMDA and muORs are strategically localized in dendrites, including those receiving excitatory synapses, of central amygdala neurons. Thus, postsynaptic co-modulation of central amygdala neurons may be a key cellular substrate mediating glutamate and opioid interaction on neural signaling and plasticity associated with normal and pathological emotional processes associated with addictive behaviors.

Cannabinoid (CB(1)), GABA(A) and GABA(B) receptor subunit changes in the globus pallidus in Huntington's disease.

Huntington's disease (HD) is a disease of the basal ganglia which results in a major loss of the striatal GABAergic medium spiny neurons containing enkephalin and substance P. These neurons project principally to the globus pallidus (GP) and substantia nigra pars reticulata (SNr). Both GABA(A) and GABA(B) receptors are localised postsynaptically on neurons in the GP and SNr, and cannabinoid (CB(1)) receptors are localised presynaptically on the axon terminals of the medium spiny projection neurons in the GP and SNr. The aims of this project were to investigate the changes in the distribution of CB(1), GABA(A), and GABA(B) receptor subunits, as well as enkephalin and substance P in the GP in the HD brain compared to the normal brain. The results of this study have shown firstly, that in the HD brain there is a dramatic loss of enkephalin and CB(1) receptor immunoreactivity (IR) in the external segment of the globus pallidus (GPe) and a major loss of substance P and CB(1) receptor-IR from the internal segment of the globus pallidus (GPi). Secondly, the degeneration of these striatal efferent neurons results in the upregulation of the various subunits of both GABA(A) (alpha(1), beta(2,3) and gamma(2)) and GABA(B) (R(1)) receptors in the GP in HD. Detailed double labelling confocal microscopy studies show that in HD the increased GABA(A) and GABA(B) receptor-IR is distributed not just in punctate "synaptic" regions, but throughout all dendritic and somal membranes of pallidal neurons. These results provide the first comprehensive description of the changes of CB(1), GABA(A) and GABA(B) receptor subunits in the HD basal ganglia. The upregulation of both GABA(A) and GABA(B) receptors may serve to increase the sensitivity of pallidal neurons to the decreased levels of GABA that occurs in the GP in HD. The loss of CB(1) receptors in HD is also thought to be a compensatory mechanism due to evidence that endocannabinoids modulate the reuptake of GABA in the GP. These findings show the high degree of plasticity of CB(1), GABA(A) and GABA(B) receptors and provide a better understanding of the GABAergic modulation of basal ganglia neurons in the normal and diseased human brain.

Altered CB1 receptor and endocannabinoid levels precede motor symptom onset in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease characterised by cell dysfunction and death in the basal ganglia and cortex. Currently there are no effective pharmacological treatments available. Loss of cannabinoid CB1 receptor ligand binding in key brain regions is detected early in HD in human postmortem tissue [Glass M, Dragunow M, Faull RL (2000) The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington's disease. Neuroscience 97:505-519]. In HD transgenic mice environmental enrichment upregulates the CB1 receptors and slows disease progression [Glass M, van Dellen A, Blakemore C, Hannan AJ, Faull RL (2004) Delayed onset of Huntington's disease in mice in an enriched environment correlates with delayed loss of cannabinoid CB1 receptors. Neuroscience 123:207-212]. These findings, combined with data from lesion studies have led to the suggestion that activation of cannabinoid receptors may be protective. However, studies suggest that CB1 mRNA may be decreased early in the disease progression in HD mice, making this a poor drug target. We have therefore performed a detailed analysis of CB1 receptor ligand binding, protein, gene expression and levels of endocannabinoids just prior to motor symptom onset (12 weeks of age) in R6/1 transgenic mice. We demonstrate that R6/1 mice exhibit a 27% decrease in CB1 mRNA in the striatum compared to wild type (WT). Total protein levels, determined by immunohistochemistry, are not significantly different to WT in the striatum or globus pallidus, but are significantly decreased by 19% in the substantia nigra. CB1 receptor ligand binding demonstrates significant but small decreases (<20%) in all basal ganglia regions evaluated. The levels of the endocannabinoid 2-arachidonoyl glycerol are significantly increased in the cortex (147%) while anandamide is significantly decreased in the hippocampus to 67% of WT. Decreases are also apparent in the ligand binding of neuronal D1 and D2 dopamine receptors co-located with CB1, while there is no change in GABA(A) receptor ligand binding. These results suggest that in this R6/1 mouse colony at 12 weeks there are only very small changes in CB1 protein and receptors and thus this would be an appropriate time point to evaluate therapeutic interventions.

Nuclear localisation of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) in invasive trophoblasts and an association with recurrent miscarriage.

Endocannabinoids are lipid signalling molecules that are related to the major psychoactive component in marijuana, delta-9-tetrahydrocannabinol and are increasingly recognized as being important in implantation and development of early embryos. The endocannabinoid anandamide, is metabolized by the enzyme fatty acid amide hydrolase (FAAH), and insufficient levels of this enzyme have been implicated in spontaneous miscarriage in women and implantation failure in mice. We screened placental bed biopsies and placental tissue from 45 women with recurrent miscarriage and 17 gestation-matched women with normal pregnancies for the expression of FAAH by immunohistochemistry. Unexpectedly, the enzyme appeared to be localised to the nucleus of trophoblasts and this was confirmed by western blotting of sub-cellular fractions and confocal microscopy. FAAH was expressed in the cytoplasm of large decidual stromal cells and significantly more women with recurrent miscarriage (73%) expressed FAAH in these cells than women with normal pregnancy (31%). FAAH was also expressed in the nucleus of extravillous trophoblasts that had invaded the decidua from 67% of women with recurrent miscarriage but was not expressed by these cells in any women with normal pregnancies. In contrast, FAAH was expressed in extravillous trophoblasts that had migrated out of the villi but that had not yet invaded the decidua in both normal pregnancies and in cases of recurrent miscarriage. FAAH was also present in the nucleus of a small number of villous trophoblasts in some specimens. FAAH appears to be over expressed in trophoblasts that have invaded the decidua, as well as in large decidual stromal cells in many cases of recurrent miscarriage. This may reflect inadequate control of the cannabinoid system in the uterus of women who experience recurrent miscarriages. The functional significance of the unexpected nuclear localisation of FAAH in trophoblasts is not yet clear.

Meta-analysis of cannabinoid ligand binding affinity and receptor distribution: interspecies differences.

A meta-analysis, unlike a literature review, synthesizes previous studies into new results. Pooled data from 211 studies measured ligand binding affinities at human (Hs) or rat (Rn) cannabinoid receptors CB1 and CB2. Cochrane methods were modified for this non-clinical analysis. Meta-regression detected data heterogeneity arising from methodological factors: use of sectioned tissues, lack of PMSF and choice of radioligand. Native brain tissues exhibited greater affinity (lower nM) than transfected cells, but the trend fell short of significance, as did the trend between centrifugation and filtration methods. Correcting for heterogeneity, mean Ki values for delta 9-tetrahydrocannabinol differed significantly between HsCB1 and RnCB1 (25.1 and 42.6 nM, respectively) but not between HsCB1 and HsCB2 (25.1 and 35.2). Mean Kd values for HsCB1, RnCB1 and HsCB2 of CP55,940 (2.5, 0.98, 0.92) and WIN55,212-2 (16.7, 2.4, 3.7) differed between HsCB1 and RnCB1 and between HsCB1 and HsCB2. SR141716A differed between HsCB1 and RnCB1 (2.9 and 1.0 nM). Anandamide at HsCB1, RnCB1 and HsCB2 (239.2, 87.7, 439.5) fell short of statistical differences due to heterogeneity. We consider these Kd and Ki values to be the most valid estimates in the literature. Sensitivity analyses did not support the numerical validity of cannabidiol, cannabinol, 2-arachidonoyl glycerol and all ligands at RnCB2. Aggregate rank order analysis of CB(1) distribution in the brain (pooled from 119 autoradiographic, immunohistochemical and in situ hybridization studies) showed denser HsCB1 expression in cognitive regions (cerebral cortex) compared to RnCB1, which was relatively richer in movement-associated areas (cerebellum, caudate-putamen). Implications of interspecies differences are discussed.

Central ventilatory control in the South American lungfish, Lepidosiren paradoxa: contributions of pH and CO(2).

Lungfish represent a probable sister group to the land vertebrates. Lungfish and tetrapods share features of respiratory control, including central, peripheral and intrapulmonary CO(2) receptors. We investigated whether or not central chemoreceptors in the lungfish, L. paradoxa, are stimulated by CO(2) and/or pH. Ventilation was measured by pneumotachography for diving animals. The fourth cerebral ventricle was equipped with two catheters for superfusion. Initially, two control groups were compared: (1) catheterized animals with no superfusion and (2) animals superfused with mock CSF solutions at pH = 7.45; PCO(2) = 21 mmHg. The two groups had virtually the same ventilation of about 40 ml BTPS kg(-1) h(-1) (P > 0.05). Next, PCO(2) was increased from 21 to 42 mmHg, while pH(CSF) was kept at 7.45, which increased ventilation from 40 to 75 ml BTPS kg(-1) h(-1). Conversely, a decrease of pH(CSF) from 7.45 to 7.20 (PCO(2) = 21 mmHg) increased ventilation to 111 ml BTPS kg(-1) h(-1). Further decreases of pH(CSF) had little effect on ventilation, and the combination of pH(CSF) = 7.10 and PCO(2) = 42 mmHg reduced ventilation to 63 ml BTPS kg(-1) h(-1).