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BIOL20922 Neuroscience RSM: Measure Ca2+ Currents

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BIOL20922 Neuroscience RSM

Questions:

1.

Researchers studying a novel compound derived from cannabis suspect that it acts via a G-protein coupled receptor to cause addiction. To assess its mechanism of action, researchers measured Ca2+currents in GABAergic neurons cultured from the ventral tegmental area of the midbrain. The figure below shows single channel recordings in the absence (A) and presence of the novel compound (B) during a step depolarization from -70 mV to +10 mV.

  1. Why do the researchers depolarise the membrane to measure Ca2+currents and what is the effect of the novel compound?       (2 marks; 5 lines)
  2.  What technique is used to measure the currents shown above, and what are the signalling mechanisms by which the compound could exert its effects? (5 marks; 12 lines)
  3. Outline an experiment to test whether the drug causes addiction in rodents. (3 marks; 8 lines)

2.

Describe, at the cellular and molecular level, what happens when a mouse whisker cell is touched to generate a sense of touch. (7 marks; 16 lines)

3.

Two cultures of heterologous cells were transfected with the human TRPA1 channel. Both cultures were treated with a dye that fluoresces with increasing calcium concentration and the fluorescence was measured over time to produce the traces in the figure below. An agonist of TRPA1, carvacrol, was applied intermittently to both cultures (black dashes below the trace). The chemical forskolin (FSK, open bar below the trace) was applied to one of the cultures.

a) Describe the action of FSK on the cellular calcium concentration.(1 mark; 2 lines)

b) TRPA1 is normally expressed in nociceptive responses. What physiological mechanism is forskolin mimicking? (3 marks; 8 lines)

c) Suggest an additional control for this experiment to confirm the specificity of the signalling pathway. (2 marks; 5 lines)

d) Outline the evidence that α-synuclein plays a causal role in the pathogenesis of Parkinson’s disease. (9 marks 20 lines)

5.

Understanding the neuronal dysfunctions in Huntington’s disease is challenging because patient samples must be derived from post-mortem tissue. To overcome these limitations and to better understand cellular pathology in Huntington’s disease, you generated induced pluripotent stem cells (iPSCs) from skin biopsies of three Huntington’s disease patients and three neurotypical healthy individuals. You then differentiated these iPSCs into medium spiny neurons (MSNs). Next, you examined huntingtin localisation in these patient-derived MSNs using two antibodies to huntingtin – Ab1 or Ab2. Your results are shown in the figure below.

[Figure legend title intentionally removed]. Representative images of MSNs derived from healthy individuals or patients with Huntington’s disease (HD). Cells were stained with either Ab1 or Ab2. Scale bar = 10 µm.

a) Based on the results above, do you consider these patient-derived neurons represent a useful model of Huntington’s disease? Justify your answer. (4 marks; 10 lines)

b) Suggest reasons why the authors achieved different results for the two anti-huntingtin antibodies? Outline an experiment to support your answer and describe the expected results. (2 marks; 10 lines)

c) You repeat the experiments using neurons derived from patients with Huntington’s disease in the absence or presence of a novel drug BIO00048I. The results are shown in the figure below.

[Figure legend title intentionally removed]. Representative images of MSNs derived from patients with Huntington’s disease. Cells were treated for 48 hours with BIO00048I (10 µM) or vehicle. Cells were stained with Ab1. Scale bar = 10 µm.

Based on your understanding of the Huntingtin protein, propose a mechanism of action for the effect of BIO00048I. Justify your answer.    (3 marks; 8 lines)

BIOL20922 Neuroscience RSM

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