Investigation of feeding behaviour in C. elegans reveals distinct pharmacological and antibacterial effects of nicotine.

Caenorhabditis elegans is an informative model to study the neural basis of feeding. A useful paradigm is one in which adult nematodes feed on a bacterial lawn which has been pre-loaded with pharmacological agents and the effect on pharyngeal pumping rate scored. A crucial aspect of this assay is the availability of good quality bacteria to stimulate pumping to maximal levels. A potential confound is the possibility that the pharmacological agent impacts bacterial viability and indirectly influences feeding rate. Here, the actions of nicotine on pharyngeal pumping of C. elegans and on the Escherichia coli bacterial food source were investigated. Nicotine caused an immediate and concentration-dependent inhibition of C. elegans pharyngeal pumping, IC50 4 mM (95% CI = 3.4 mM to 4.8 mM). At concentrations between 5 and 25 mM, nicotine also affected the growth and viability of E. coli lawns. To test whether this food depletion by nicotine caused the reduced pumping, we modified the experimental paradigm. We investigated pharyngeal pumping stimulated by 10 mM 5-HT, a food 'mimic', before testing if nicotine still inhibited this behaviour. The IC50 for nicotine in these assays was 2.9 mM (95% CI = 3.1 mM to 5.1 mM) indicating the depletion of food lawn does not underpin the potency of nicotine at inhibiting feeding. These studies show that the inhibitory effect of nicotine on C. elegans pharyngeal pumping is mediated by a direct effect rather than by its poorly reported bactericidal actions.

Selective and compartmentalized myelin expression of HspB5.

In the present study, we reveal myelin-specific expression and targeting of mRNA and biochemical pools of HspB5 in the mouse CNS. Our observations are based on in situ hybridization, electron microscopy and co-localization with 2',3'-Cyclic-Nucleotide 3'-Phosphodiesterase (CNPase), reinforcing this myelin-selective expression. HspB5 mRNA might be targeted to these structures based on its presence in discrete clusters resembling RNA granules and the presence of a putative RNA transport signal. Further, sub-cellular fractionation of myelin membranes reveals a distinct sub-compartment-specific association and detergent solubility of HspB5. This is akin to other abundant myelin proteins and is consistent with HspB5's association with cytoskeletal/membrane assemblies. Oligodendrocytes have a pivotal role in supporting axonal function via generating and segregating the ensheathing myelin. This specialization places extreme structural and metabolic demands on this glial cell type. Our observations place HspB5 in oligodendrocytes which may require selective and specific chaperone capabilities to maintain normal function and neuronal support.

Alpha-synuclein deficiency in the C57BL/6JOlaHsd strain does not modify disease progression in the ME7-model of prion disease.

We previously detailed how intrahippocampal inoculation of C57BL/6J mice with murine modified scrapie (ME7) leads to chronic neurodegeneration (Cunningham C, Deacon R, Wells H, Boche D, Waters S, Diniz CP, Scott H, Rawlins JN, Perry VH (2003) Eur J Neurosci 17:2147-2155.). Our characterization of the ME7-model is based on inoculation of this murine modified scrapie agent into C57BL/6J mice from Harlan laboratories. This agent in the C57BL/6J host generates a disease that spans a 24-week time course. The hippocampal pathology shows progressive misfolded prion (PrP(Sc)) deposition, astrogliosis and leads to behavioural dysfunction underpinned by the early synaptic loss that precedes neuronal death. The Harlan C57BL/6J, although widely used as a wild type mouse, are a sub-strain harbouring a spontaneous deletion of alpha-synuclein with the full description C57BL/6JOlaHsd. Recently alpha-synuclein has been shown to ameliorate the synaptic loss in a mouse model lacking the synaptic chaperone CSP-alpha. This opens a potential confound of the ME7-model, particularly with respect to the signature synaptic loss that underpin the physiological and behavioural dysfunction. To investigate if this strain-selective loss of a candidate disease modifier impacts on signature ME7 pathology, we compared cohorts of C57BL/6JOlaHsd (alpha-synuclein negative) with the founder strain from Charles Rivers (C57BL/6JCrl, alpha-synuclein positive). There were subtle changes in behaviour when comparing control animals from the two sub-strains indicating potentially significant consequences for studies assuming neurobiogical identity of both strains. However, there was no evidence that the absence of alpha-synuclein modifies disease. Indeed, accumulation of PrP(Sc), synaptic loss and the behavioural dysfunction associated with the ME7-agent was the same in both genetic backgrounds. Our data suggest that alpha-synuclein deficiency does not contribute to the compartment specific processes that give rise to prion disease mediated synaptotoxicity and neurodegeneration.

A randomized controlled trial of treatment options for troublesome uterine bleeding in Implanon users.

BACKGROUND: Pilot data have indicated that both doxycycline alone and mifepristone combined with ethinyl estradiol (EE) are effective in stopping episodes of bleeding in Implanon users with troublesome bleeding. We compared four treatments against a placebo in Implanon users and tested whether repeated treatment improved subsequent bleeding patterns. METHOD: Implanon users aged 18-45 years were randomized to treatment with (i) mifepristone 25 mg given twice on day 1 followed by 4 days of EE 20 microg; (ii) doxycycline 100 mg twice daily for 5 days; (iii) mifepristone 25 mg given twice on day 1 plus doxycycline 100 mg twice daily for 5 days; (iv) doxycycline 100 mg twice daily with EE 20 microg daily; and (v) placebo twice daily for 5 days. The primary end-point was the number of days of bleeding/spotting immediately following initiation of the first 5-day course of each therapy, compared with placebo. RESULTS: There were 204 women assigned to treatment. Mifepristone in combination with either EE or doxycycline was significantly more effective in stopping an episode of bleeding (mean 4.0 days (CI 3.5-4.6) and 4.4 days (CI 3.8-5.2), respectively) than doxycycline alone or in combination with EE, or placebo (6.4 days (CI 4.4-9.2), 6.4 days (CI 4.8-8.6) and 6.4 days (CL 5.1-8.0), respectively). CONCLUSION: Mifepristone combined with either EE or doxycycline was significantly more effective than placebo in terminating an episode of bleeding in Implanon users. However there was no improvement in subsequent bleeding patterns. TRIAL REGISTRATION NUMBER: ACTR # 012605000206628.

Expression of the small heat shock protein family in the mouse CNS: differential anatomical and biochemical compartmentalization.

The small heat shock proteins (sHsps) are a family of molecular chaperones defined by an alpha-crystallin domain that is important for sHsps oligomerization and chaperone activity. sHsps perform many physiological functions including the maintenance of the cellular cytoskeleton, the regulation of protein aggregation and modulate cell survival in a number of cell types including glial and neuronal cells. Many of these functions have been implicated in disease processes in the CNS and indeed sHsps are considered targets for disease therapy. Despite this, there is no study that systematically and comparatively characterized sHsps expression in the CNS. In the present study we have analyzed the expression of this gene family in the mouse brain by reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting. Gene expression analysis of the 10 known members of mammalian sHsps confirms the presence of 5 sHsps in the CNS. A distinct white matter specific expression pattern for HspB5 and overlapping expression of HspB1 and HspB8 in the lateral and dorsal ventricles of the brain is observed. We confirm protein expression of HspB1, HspB5, HspB6 and HspB8 in the brain. Further subcellular fractionation of brain and synaptosomes details a distinct subcompartment-specific association and detergent solubility of sHsps. This biochemical signature is indicative of an association with synaptic and other neural specializations. This observation will help one understand the functional role played by sHsps during physiology and pathology in the CNS.

Mobility in patients with venous leg ulceration.

OBJECTIVES: To compare mobility in patients with venous leg ulcers to matched controls and determine the influence of mobility, age and ulcer size on ulcer healing. METHODS: 25 leg ulcer patients, and 25 matched controls wore a mobility monitor (ActivPAL, PAL Technologies Ltd, Glasgow, Scotland)) which recorded the number of steps and amount of time spent walking, standing, sitting or lying for a one-week period. A walking index was calculated. The ulcer group were treated with compression bandaging and ulcer healing recorded over 12 weeks. RESULTS: There were 13 female subjects in each group. The median age was 70.5 (range 30-89) years. There was no difference in the amount of time either group spent standing, walking and resting. There was a significant reduction in the number of steps taken and in the walking index in the ulcer group compared to controls (ulcer group, median 6,685 steps/day, range 2074-17,999; control group median 8750, range 4917-16,043, p<0.05, Mann Whitney u test). Smaller ulcers and ulcers of recent onset were most likely to heal within 12 weeks (p=0.005 and p=0.011 respectively, Chi squared test). The percentage of time spent mobilising and resting did not influence ulcer healing (r(s)=-0.125; p=0.55). CONCLUSIONS: Mobility patterns among patients with leg ulcers are not significantly different to age matched controls. Ulcer patients take fewer steps per week compared to controls indicating they have reduced calf muscle pump function. Further studies are required to determine whether therapies which increase calf muscle activity have a role in ulcer treatment.

The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.

The effects of ethanol on the brain are concentration dependent. Low concentrations (mM) intoxicate, while greater than 100 mM anaesthetize. Of most relevance to human alcohol addiction are mechanisms of intoxication. Previously, Caenorhabditis elegans has been employed in genetic screens to define effectors of intoxication. Here, we inform interpretation of these studies by providing evidence that ethanol rapidly equilibriates across C. elegans cuticle. Importantly, the effect of ethanol on muscle activity rapidly reaches steady-state, and the concentration-dependence of the effect is very similar in intact animals and exposed muscle. Thus the cuticle does not present an absorption barrier for ethanol, and furthermore the internal concentration is likely to approach that applied externally. Thus, modelling intoxication in C. elegans requires exposure to external ethanol less than 100 mM. Furthermore, the permeability of the cuticle to ethanol enables analysis of precisely controlled concentration-dependent effects of acute, chronic, and episodic ethanol exposure on behaviour.

Molecular characterization of the metabotropic glutamate receptor family in Caenorhabditis elegans.

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that play an important neuromodulatory role in the brain. Glutamatergic transmission itself plays a fundamental role in the simple nervous system of the model organism Caenorhabditis elegans, but little is known about the contribution made by mGluR signalling. The sequenced genome of C. elegans predicts three distinct genes, mgl-1, mgl-2 and mgl-3 (designated Y4C6A.2). We have used in silico and cDNA analyses to investigate the genes encoding mgls. Our results indicate that mgl genes constitute a gene family made up of three distinct subclasses of receptor. Our transcript analysis highlights potential for complex gene regulation with respect to both expression and splicing. Further, we identify that the predicted proteins encoded by mgls harbour structural motifs that are likely to regulate function. Taken together, this molecular characterization provides a platform to further investigate mGluR function in the model organism C. elegans.

Role of NSF in neurotransmitter release: a peptide microinjection study at the crayfish neuromuscular junction.

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 +/- 12% (mean +/- SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.

Molecules of the mind: integrating synaptic biochemistry to understand brain function.

The focused meeting entitled 'Molecular Determinants of Synaptic Function: Molecules and Models' brought together several molecules and experimental models that are furthering our understanding of the biochemical basis of integrative brain function. Invited speakers and short communications from more junior scientists highlighted how individual molecules or protein networks underlie defined subcellular functions (e.g. transmitter release, receptor activation and transmitter uptake) can be used to unravel integrative function at cellular, systems and behavioural levels.

Increased expression of glial fibrillary acidic protein fragments and mu-calpain activation within the hippocampus of prion-infected mice.

Prion diseases are characteristically accompanied by marked astrocytic activation, which is initiated relatively early in the disease process. Using the intracerebrally injected ME7 strain of prion agent to model disease, we identified an expected increase in GFAP (glial fibrillary acidic protein) but additionally noted an accumulation of GFAP cleavage fragments in hippocampal homogenates. A time-dependent increase in hippocampal mu-calpain immunoreactivity within astrocytes suggests that its proteolytic activity may account for the cleavage of GFAP that is observed in the ME7 model. It may therefore contribute to the reactive gliosis that is characteristic of prion diseases.

A pilot study to assess the effect of three short-term treatments on frequent and/or prolonged bleeding compared to placebo in women using Implanon.

BACKGROUND: The major side-effect of progestogen-only contraception is disruption of menstrual bleeding patterns, which can lead to a high incidence of early discontinuation. The aim of this study was to compare three treatments with placebo on the duration and recurrence of frequent and/or prolonged bleeding in Implanon users. METHOD: Women between the ages of 18 and 45 years, who had used Implanon for > or =3 months and were experiencing prolonged or frequent bleeding patterns, were recruited at four Australian sites. Subjects were randomized to treatment using computer-generated random number table if they met the World Health Organization criteria for prolonged and/or frequent bleeding in the previous 90 days [Belsey, E.M., Pinol, A.P.Y. and Taskforce on Long-Acting Systemic Agents for Fertility Regulation, World Health Organization (1997) Contraception 55,57-65]. Treatments were: (1) mifepristone 25 mg given twice on day 1 followed by 4 days of twice daily placebo; (2) mifepristone 25 mg given twice on day 1 followed by 4 days of ethinyl estradiol (EE) 20 microg in the morning and placebo at night; (3) doxycycline 100 mg twice daily for 5 days; and (4) placebo twice daily for 5 days. Analysis was by intention to treat. The primary endpoint was the number of days of bleeding and spotting immediately following initiation of the 5 day course of each active therapy compared with placebo. RESULTS: A total of 179 women was assigned to treatment. Both mifepristone in combination with EE and doxycycline alone were significantly more effective in stopping an episode of bleeding {mean 4. 3 days [confidence interval (CI) 3.5-5.2], and 4.8 days (CI 3.9-5.8) respectively} than mifepristone alone or placebo [5.9 days (CI 4.8-7.2) and 7.5 days (CI 6.1-9.1) respectively]. No effect on subsequent bleeding patterns was observed in any treatment group. CONCLUSION: Both mifepristone plus EE and doxycycline alone were significantly more effective than placebo in terminating an episode of bleeding in women with prolonged and/or frequent bleeding using Implanon. We believe that the observed reduction in the number of bleeding days by almost 50% compared to placebo in both the mifepristone combination group and the doxycycline group demonstrates a clinically significant improvement in bleeding patterns and that further trials are needed to compare different combinations of therapy as well as multiple dosing regimens in order to establish which is the most effective treatment option. The effect of repeat administration or combinations of these preparations on long-term bleeding patterns requires further investigation.

Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods.

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission. The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6-8) mGluRs. In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3. Prominent among the candidate receptor interacting proteins was calmodulin, a Ca(2+) sensor known to bind identifiable sequences in group I and III mGluRs. The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs. Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs. This illustrates that certain Ca(2+)-dependent interactions are not recapitulated in yeast. Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.

Eml5, a novel WD40 domain protein expressed in rat brain.

We have isolated a novel transcript with homology to the major microtubule-associated protein in dividing sea urchin embryos, EMAP. The protein has a predicted MW of approximately 180 kDa and we have named it Eml5 (EMAP-like protein 5). Eml5 contains 11 putative WD40 domains and 3 hydrophobic stretches of 43 aa, HELP domains, which have been suggested to be involved in microtubule binding. Eml5 appears to consist of two tandem repeats of the complete EMAP protein separated by a putative dimerization domain. Eml5 mRNA and protein is expressed at high levels in the hippocampus, cerebellum and olfactory bulb, as determined by in situ hybridization and immunocytochemistry. Eml5 transcripts can be detected in fore- and hindbrain structures from embryonic day 13 onwards. Because other EMAP-like proteins are involved in regulating microtubule dynamics, it is likely that Eml5 plays a role in the regulation of cytoskeletal rearrangements during neuronal development and in adult brain

LTP but not seizure is associated with up-regulation of AKAP-150.

We have used differential display to profile and compare the mRNAs expressed in the hippocampus of freely moving animals after the induction of long-term potentiation (LTP) at the perforant path-dentate gyrus synapse with control rats receiving low-frequency stimulation. We have combined this with in situ hybridization and have identified A-kinase anchoring protein of 150 kDa (AKAP-150) as a gene selectively up-regulated during the maintenance phase of LTP. AKAP-150 mRNA has a biphasic modulation in the dentate gyrus following the induction of LTP. The expression of AKAP-150 was 29% lower than stimulated controls 1 h after the induction of LTP. Its expression was enhanced 3 (50%), 6 (239%) and 12 h (210%) after induction, returning to control levels by 24 h postinduction. The NMDA receptor antagonist CPP blocked the tetanus-induced modulation of AKAP-150 expression. Interestingly, strong generalized stimulation produced by electroconvulsive shock did not increase the expression of AKAP-150. This implies that the AKAP-150 harbours a novel property of selective responsiveness to the stimulation patterns that trigger NMDA-dependent LTP in vivo. Its selective up-regulation during LTP and its identified functions as a scaffold for protein kinase A, protein kinase C, calmodulin, calcineurin and ionotropic glutamate receptors suggest that AKAP-150 encodes is an important effector protein in the expression of late LTP.

Ntab, a novel non-coding RNA abundantly expressed in rat brain.

We have identified a novel transcript that is abundantly and specifically expressed in both the adult and developing rat CNS. Within the full-length cDNA sequence we were unable to identify a clear open reading frame. Moreover, we were unable to detect any protein product derived from the full-length cDNA sequence using an in vitro translation assay. Therefore, we suggest this gene is one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as an RNA. We have named this novel gene Ntab for non-coding transcript abundantly expressed in brain (accession number AY035551). In addition, in some regions of the brain we find evidence for RNA accumulation in cellular processes at some distance from the soma. These findings suggest that Ntab is actively transported and may function within cellular processes. Since Ntab is a targeted non-coding RNA, such cellular functions could include the targeting and/or regulation of localised translation of other mRNA species.

Mapping of calmodulin and Gbetagamma binding domains within the C-terminal region of the metabotropic glutamate receptor 7A.

Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades.

Subfield-specific immediate early gene expression associated with hippocampal long-term potentiation in vivo.

It is not known whether NMDA receptor-dependent long-term potentiation (LTP) is mediated by similar molecular mechanisms in different hippocampal areas. To address this question we have investigated changes in immediate early gene and protein expression in two hippocampal subfields following the induction of LTP in vivo and in vitro. In granule cells of the dentate gyrus, LTP induced in vivo by tetanic stimulation of the perforant path was followed by strong induction of the immediate early genes (IEGs) Zif268, Arc and Homer. The increase in Zif268 mRNA was accompanied by an increase in protein expression. In contrast, we were unable to detect modulation of the IEGs Zif268, Arc, Homer and HB-GAM following induction of LTP by high-frequency stimulation of the commissural projection to CA1 pyramidal cells in vivo. In this pathway, we also failed to detect modulation of Zif268 protein levels. Zif268, Arc and Homer can be modulated in CA1 pyramidal cells approximately twofold after electroshock-induced maximal seizure, which demonstrates potential responsiveness to electrical stimuli. When LTP was induced in vitro neither CA1 pyramidal cells nor granule cells showed an increase in Zif268, Arc or Homer mRNA. However, in the slice preparation, granule cells have a different transcriptional state as basal IEG levels are elevated. These results establish the existence of subfield-specific transcriptional responses to LTP-inducing stimulation in the hippocampus of the intact animal, and demonstrate that in area CA1-enhanced transcription of Zif268, Arc and Homer is not required for the induction of late LTP.

Seizure-induced gene expression in area CA1 of the mouse hippocampus.

Synaptic plasticity in the hippocampus requires activity-dependent gene expression. We have therefore profiled gene expression in area CA1 following the induction of an electroshock-evoked maximal seizure. Using cDNA microarrays, the differential expression of approximately 9000 cDNAs was examined. In situ hybridization on 14 transcripts that showed strongest modulation in the microarray screen (1.8-2-fold) confirmed the differential expression of a single gene that encodes for the nuclear hormone receptor NGFI-B (Nur77, N10). Although this gene is only modestly up-regulated (approximately 2-fold) in area CA1, in situ hybridization revealed that maximal seizures induce a marked (approximately 12-fold) up-regulation of NGFI-B in the dentate gyrus. These data support the notion [French et al. (2001) Eur. J. Neurosci., 13, 968-976] that CA1 pyramidal neurons are more refractory than granule cells of the dentate gyrus with respect to activity-dependent gene transcription. Furthermore, our results argue against a large cohort of activity-dependent genes in area CA1.

Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling.

Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.