Why study thermal sensation?

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• Why study thermal sensation?
• 1. Most recent review 1986!
• 2. Kandel on thermal sensation
• very little, only 2 figs
• compare with e.g. ear, eye, smell, taste
• little detail - how does transduction work?
• - the question is not even asked..... This is a
  strong clue!

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Kandel on thermoreceptors
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In contrast - Kandel on vision
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In the beginning cold spots identified
by Magnus Blix
Norrsell et al Brain Res Bull 1999
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Norrsell et al Brain Res Bull 1999
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Sensory spots on Blixs left wrist
warm
cold
touch
Norrsell et al Brain Res Bull 1999
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Underneath the sensory spots......
From Kandel et al. Principles of Neural Science
3rd ed. (1991)
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How to get at the membrane? - use the soma as a
model of the terminal
In vivo.
Modified from Kandel et al. Principles of Neural
Science 3rd ed. (1991)
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How to get at the membrane? - use the soma as a
model of the terminal
In culture.
No axon, so channels appear in the soma
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How to get at the membrane? - use the soma as a
model of the terminal
In culture.
No axon, so channels appear in the soma
Useful points ? The soma is accessible so ? Can
apply Ca2 imaging ? Can use all varieties of
patch clamping
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How to get at the membrane? - use the soma as a
model of the terminal
In culture.
No axon, so channels appear in the soma
Useful points ? The soma is accessible so ? Can
apply Ca2 imaging ? Can use all varieties of
patch clamping
Problems ? Mixture of channels from
terminal, soma and axon ? Change of phenotype in
culture
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Noxious heat activates some cultured DRG neurones
(Cesare McNaughton PNAS 1996Cesare et al PNAS
1997)
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....by way of the capsaicin receptor TRPV1 (VR1)
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Cold sensingin theDRG model
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Pre-selection of cold-sensitive DRG neurones
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Pre-selection of cold-sensitive DRG neurones
Reid, Babes Pluteanu J Physiol 2002
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Pre-selection of cold-sensitive DRG neurones
Reid, Babes Pluteanu J Physiol 2002
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Pre-selection of cold-sensitive DRG neurones
Reid, Babes Pluteanu J Physiol 2002
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Pre-selection of cold-sensitive DRG neurones
Reid, Babes Pluteanu J Physiol 2002
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Pre-selection of cold-sensitive DRG neurones
Reid, Babes Pluteanu J Physiol 2002
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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How does cooling affect these neurones?
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Cold- and menthol-activated current
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Cold- and menthol-activated current
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Initial questions on thecold- and
menthol-activated current
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Initial questions on thecold- and
menthol-activated current

• does it explain cold transduction in real cold
  receptors?

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Initial questions on thecold- and
menthol-activated current

• does it explain cold transduction in real cold
  receptors?
• do cold and menthol activate the ion channel
  directly or indirectly?

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Initial questions on thecold- and
menthol-activated current

• does it explain cold transduction in real cold
  receptors?
• do cold and menthol activate the ion channel
  directly or indirectly?

- what properties of intact cold receptorsare
accounted for by this current?
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Initial questions on thecold- and
menthol-activated current

• does it explain cold transduction in real cold
  receptors?
• do cold and menthol activate the ion channel
  directly or indirectly?

- what properties of intact cold receptorsare
accounted for by this current?
- how does it behave in excised patches?
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What properties of cold receptors does the
current account for?(1) Cold and menthol effects
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(1) Cold and menthol effects
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(1) Cold and menthol effects
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(1) Cold and menthol effects
...very likely that (menthol) exerts its
actionupon an enzyme...concerned in the
thermallyconditioned regulation of the discharge
ofthe cold receptors (Hensel Zotterman 1951)
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(2) Adaptation
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What properties of cold receptors does the
current account for?(2) Adaptation
Reid Flonta Nature 2001
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What properties of cold receptors does the
current account for?(3) Modulation by calcium
Low Ca2o stimulates cold receptors (Schäfer et
al J Neurophysiol 1982)
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What properties of cold receptors does the
current account for?(3) Modulation by calcium
Low Ca2o stimulates cold receptors (Schäfer et
al J Neurophysiol 1982)
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(3) Modulation by calcium
High Ca2o antagonises the actionof
menthol (Schäfer et al J Gen Physiol 1986)
Low Ca2o stimulates cold receptors (Schäfer et
al J Neurophysiol 1982)
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(3) Modulation by calcium
High Ca2o antagonises the actionof
menthol (Schäfer et al J Gen Physiol 1986)
Low Ca2o stimulates cold receptors (Schäfer et
al J Neurophysiol 1982)
Reid, Babes Pluteanu J Physiol 2002
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What properties of cold receptors does the
current account for?(3) Modulation by calcium
High Ca2o antagonises the actionof
menthol (Schäfer et al J Gen Physiol 1986)
Low Ca2o stimulates cold receptors (Schäfer et
al J Neurophysiol 1982)
Reid, Babes Pluteanu J Physiol 2002
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Is the channel directly activated by cold and
menthol?
i.e. does it still function inexcised
outside-out patches?
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Yes, it does......
No menthol
100 µM menthol
Reid Flonta Neurosci Lett 2002
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Yes, it does......but......
No menthol
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...what about its cold sensitivity?
Patch 100 mM menthol
Raw current 100 mM menthol
Summed n 10
Whole cell 100 mM menthol
Reid Flonta Neurosci Lett 2002
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...they are nonselective cation channels
From Reid Flonta Neurosci Lett 324164-168
(2002)
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The cold and menthol receptor

• Cold and menthol directly activate a nonselective
  cation channel
• This channel can account for
• cold sensing
• sensitisation by menthol
• adaptation
• sensitisation by low Ca2o
• antagonism of menthol response by high Ca2o
• i.e. innocuous cold transduction,and all known
  effects of menthol and calciumon cold receptors
  in vivo
• It is probably a major mechanism of innocuous
  cold sensing

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Answers to some of your questions about TRPM8
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Stereospecificity of menthol action
From Reid, Babes Pluteanu J Physiol 545595-614
(2002)
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Pathways of Ca2 entry
From Reid, Babes Pluteanu J Physiol 545595-614
(2002)
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Pathways of Ca2 entry
From Reid, Babes Pluteanu J Physiol 545595-614
(2002)
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The cold-induced depolarisation and currentare
largely Na-dependent
From Reid, Babes Pluteanu J Physiol 545595-614
(2002)
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Cloning of the cold and menthol receptor TRPM8
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Cloning of the cold and menthol receptor TRPM8
Expression cloning cDNA library from trigeminal
ganglion, screened with Ca2 imaging using high
concentration of menthol McKemy et al. Nature
41652-58 (7 Mar 2002)
Bioinformatics approach search of genomic
databases for sequences with similarity to TRP
channels, followed by RACE PCR to get full-length
sequence Peier et al. Cell 108705-715 (8 Mar
2002)
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Properties of TRPM8
Cold and menthol responsive
Nonselective cation channel
From McKemy et al Nature 41652-58 (2002)
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TRPA
Clapham, Nature 2003
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Temperature-sensitive TRP channels
TRPM8 TRPV3 TRPV1 (VR1)
TRPV2 (VRL1)
Modified from McKemy et al Nature 41652-58 (2002)
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TRPM8 is expressed in small TG/DRG neurones
6 kb
4.5 kb
Cyclophilin
50 µm
From McKemy et al Nature 41652-58 (2002)
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How does adaptation happen?
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Adaptation depends on Ca2 entry
Reid, Babes Pluteanu J Physiol 2002
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Adaptation involves a shift in cold sensitivity
Reid, Babes Pluteanu J Physiol 2002
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...which can be induced by raising Ca2i even
without cooling
Reid, Babes Pluteanu J Physiol 2002
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