An investigation into the role of cannabinoid and group 1 metabotropic glutamate receptors in the control of rhythmic locomotion in hatchling Xenopus Laevis

  • Jack Gibson

Student thesis: Doctoral Thesis


The group 1 metabotropic glutamate receptors (mGluR1/5) are of key interest in the synaptic plasticity that underlies learning and memory and have been implicated as a cause or target in many disease models such as Parkinson’s, Huntington’s, Alzheimer’s, depression, schizophrenia and fragile X syndrome. Alongside this, group 1 mGluRs are linked, via the production endocannabinoid 2-archidonyl glycerol (2-AG), to cannabinoid receptor 1 (CB1). CB1 is one of the most highly expressed G-protein coupled receptors (GPCRs) in the central nervous system (CNS) and its function has not been confirmed in Xenopus laevis tadpoles. Debate and research are still ongoing as to how CB1 can be targeted to improve disease outcomes and the extent of its effect in vivo.

The pattern of neuronal signalling in the X. laevis central pattern generator (CPG) has been well categorised using electrophysiological recording in the immobilised tadpole at stages 37-42. We sought to build on these previous experiments to assess the swimming behaviour of the X. laevis tadpole in vivo using slow-motion high frame rate video (400 frames per second) to determine the frequency of tail swim cycles and the angle of tail flexion achieved during these swim cycles. Using this well-characterised model the effects of the two GPCRs can be determined in the unadulterated swimming behaviour. X. laevis tadpoles are also advantageous because of low cost in maintenance, a simple primary culture that can be set up at room temperature and an ectothermic development meaning large batches can be staggered in age. We looked to assess well-known plasticity inducing modulatory receptors mGluR1/5 and corroborate electrophysiological recordings previously showing mGluR1/5 activation via Dihydroxyphenylglycine (DHPG) increased motoneuron output frequency. We tested if, as in other models such as lamprey, this increase is partly mediated by retrograde cannabinoid signalling.

Our results show group 1 metabotropic glutamate receptors cause increases in the frequency of X. laevis swim-cycles at stage 40-42 evidenced through the application of DHPG, a group 1 mGluR agonist. This increase appears to be mainly through mGluR1 as inhibition of this receptor via LY367385 caused significant decreases in swim cycle frequency whereas inhibition of mGluR5 with 2-Methyl-6-(phenylethynyl)pyridine (MPEP) caused no significant decreases, indicating an intrinsic role for mGluR1 over mGluR5 in the maintenance of normal swim cycle frequency. Antagonism of CB1 with AM-251 caused significant decreases at 50µM and 10μM. However, a significant increase was observed at 2μM indicating a biphasic effect dependent on concentration. Inhibition of CB1 with AM-251 (10μM) followed by application of DHPG (50μM) had no significant effect on the frequency of swim cycles when compared with vehicle control, indicating that the increase seen with DHPG application may be blocked by CB1 inhibition. Application of endogenous CB1 agonist N-arachidonoylethanolamide (AEA) decreased frequency of swim cycles at lower concentrations (0.1-10μM), but no significant change was observed at the highest concentration (50μM). This may be some form of partial antagonism due to a higher affinity of AEA than other endogenous ligand 2-AG, but lower efficacy, effectively occupying the CB1 receptor without activation. Our data suggest that mGluR5 and CB1 may be involved in the normal muscle flexion during swimming with the application of MPEP alone or with AM-251 causing significant decreases in the angle of tail flexion.

We wanted to see if these changes in behaviour reflect in the morphology of neurons, particularly the dendritic spines dimension and membrane viscoelasticity. To do this, a primary X. laevis neuron-muscle co-culture was developed from the literature. The cultures were treated with the same pharmacological treatments and fixed in glutaraldehyde 5%. The dendritic spines of the neurons were scanned with an Atomic Force Microscope (AFM). From these scans the dendritic spine dimensions measured were; radius, volume, cross-sectional area, and membrane roughness. Using the phase contrast images, the loss tangent was calculated to give a unitless ratio of membrane stiffness. This measure of the stiffness/viscosity of the sample was used to determine if the dendritic spine head changed significantly in stiffness after group 1 mGluR or CB1 targeted drug treatment.

With AFM analysis of dendritic spine morphology and membrane stiffness, we found that group 1 mGluR activation elongated dendritic spines corroborating previous evidence in immature developing spines and in mature hippocampal cultures. Interestingly inhibition of mGluR5 also elongated the spines alongside increasing the volume of the spines. Both mGluR5 inhibition with MPEP and group 1 mGluR activation with DHPG caused significant increases in dendritic spine membrane stiffness compared with vehicle controls.

We aimed to investigate if the 20-minute application of treatments induced changes in protein expression, particularly postsynaptic density protein-95 (PSD-95) a postsynaptic structural protein present in glutamate synapses linked to NMDAR function, a key receptor in the glutamate excitation of descending interneurons (dINs).

After the behavioural assessment, the tadpoles were fixed and embedded in paraffin wax for microtome sections, which enabled observation of the spinal cord, which was investigated for changes in PSD-95 density using immunohistochemical means. After scanning with AFM, the cultures were stained with anti-PSD-95 antibodies and changes in fluorescence were measured. The results of this investigation were inconclusive due to large autofluorescence meaning a clear positive signal could not be identified.

The in vivo swimming analysis gives almost completely unadulterated tadpoles for analysis of swim-cycle output with a very well understood neuronal network that serves as an excellent model for the interplay between excitatory and inhibitory signalling of CPG networks.

In future AFM scanning can be used alongside fluorescent confocal microscopy to build a detailed picture of morphology and changes in membrane dynamics that may aid our understanding of synapse formation in normal development and in genetic disease such as fragile X.
Date of Award15 Oct 2020
Original languageEnglish
Awarding Institution
  • Abertay University
SupervisorAnne Savage (Supervisor), Nia A. White (Supervisor) & Pete Moult (Supervisor)

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