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NROC61 - Sutcliffe article notes

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Le Boutillier

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-neuropeptide system, centered in dorsolateral hypothalamus, that regulates arou
sal states, influences
feeding, and plays a role in sleep disorder narcolepsy
-hypocretin aka orexin system: also influences hormone secretion and autonomic h
-most human narcolepsies result from autoimmune attack against hypocretin-produc
ing neurons
-discovery of a neuropeptide system that regulates arousal states and energy met
-hypocretins aka orexins are 2 carboxy-terminally amidated neuropeptides of rela
ted sequence
-they're produced from a common precursor, which is expressed only in a few thou
sand neurons of rat
dorsolateral hypothalamus
-2 G protein coupled receptors (GPCRs) for hypocretins have been id'd, they have
diff distrubution w/i CNS
and diff affinities for the 2 hypocretins
-hypocretins (H) detected in secretory vesicles at synapses of fibers that proje
ct to areas w/i posterior
hypothalamus that are implicated in feeding behaviours & hormone secretion
-H fibers project to diverse targets in brain and spinal cord, incl areas implic
ated in cardiovascular
function and sleep-wake regulation
-peptides are excitatory when applied directly in vivo, and to cultures neurons
& slices in vitro, also
evidence for some inhibitory signalling
-admin of H stimulates food intake, affects blood pressure, hormone secretion, a
nd locomotor activity, and
inc wakefulness while suppressing REM sleep
-inactivating mutations in gene for H receptor 2 (Hcrtr2) in dogs = narcolepsy
-mice w/ inactivated preprohypocretin (Hcrt) gene = narcolepsy-like phenotype
-humans w/ narcolepsy have greatly reduced levels of H peptides in their CSF, an
d almost no H neurons in their
hypothalami = autoimmune attack against H neurons
-one aspect of H activity is direct excitation of cholinergic forebrain neurons
and brainstem monoaminergic
REM-off neurons in locus coeruleus, dorsal raphe nucleus and tuberomammillary nu
cleus (TMN) which together
suppress SWS
-H modulate activity of cholinergic REM-on neurons in brainstem, which gate REM
-dominant activities of H system are maintenace of waking state and suppression
of REM entry
Discovery and properties of H
-1st glimpse of H came from open-system search for undiscovered homeostatic regu
latory peptides
Analysis of all the mRNAs that are expressed by a tissue without regard to wheth
er the mRNAs have previously
been identified. The contrasting approach is to measure the expression of known
mRNAs, as is done in
cDNA array (chip) studies.
-systematic subtractive hybridization survey to id mRNA species expressed only i
n discrete nuclei w/i rat

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-40% of mRNA species that were highly enriched in hypothalamus encoded hormones
& releasing factors
-among new mRNAs was a species w/ a pattern of expression (detected by in situ h
ybrid.) restricted to a few
thousand neurons that were bilaterally distributed w/i dorsolateral hypothalamus
-the complementary DNA sequences of the rat and homologous mouse mRNAs encoded a
130-residue, putative
secretory protein w/ an apparent signal sequence
-features indicated that the product of this hypothalamic mRNA was a preprohormo
ne for 2 carboxy-terminally
amidated, secreted peptides
-one was hypocretin 2 (Hcrt2), predicted to contain 28 residues
-other was hypocretin 1 (Hcrt1), undefined amino-terminal extent
-carboxy-terminal 19 residues of the 2 peptides shared 13 AA identities, thus th
ey had related structures
and functions -this region of Hcrt2 had 7 AA match w/ secretin
-detection of H peptides w/i brain allowed their structures to be determined by
mass spectrometry
-Hcrt1 has 2 intrachain disulphide bonds
-human HCRT1 is identical to the rodent peptide, whereas human HCRT2 differs fro
m rodent Hcrt2 at 2 residues
-genes that encode preprohypocretins in pufferfish and frogs, so gene arose earl
y in chordate lineage
-sequence similarities w/ members of incretin family, esp secretin = Hcrt gene f
ormed from secretin gene
by 3 genetic rearrangements:
1) duplication of secretin gene
2) secretin w/ amino & carboxyl termini circularly permuted
3) further duplication of permuted gene w/ modifications to form gene that encod
ed 2 related peptides
-portions of 3D solution structures of hypocretins and secretin are similar (det
ermined by nuclear magnetic
resonance) despite their primary sequence consisting of 2 adjacent alpha helices
(6-7 and 9-14 AA in length)
separated by 2-3 AA turn, longer helix = region of identity btwn the 2 peptides
The H receptors
-initial orphan GPCR, (Hcrtr1) bound Hcrt1 w/ high affinity, but Hcrt2 w/ 100-10
00 fold lower affinity
-related GPCR, (Hcrtr2) had high affinity for Hcrt2 and Hcrt1
-mRNAs that encode for the 2 H receptors are enriched in brain and abundant in h
ypothalamus, but had diff
distribution in brain
-Hcrtr1 mRNA prominent in prefrontal & intralimbic cortex, hippocampus, paravent
ricular thalamic nucleus,
ventromedial hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus
-Hcrtr2 mRNA detected in cerebral cortex, septal nuclei, hippocampus, medial tha
lamic groups, raphe nuclei,
various nuclei of hypothalamus, incl TMN, dorsomedial nucleus, paraventricular n
ucleus and
ventral premammillary nucleus
Localization of the H
-hypothalamus is ancient region of mammalian brain, is a collection of distinct,
autonomously active nuclei
-several of these nuclei are regulatory centers for autonomic and endocrine home
ostatic systems
-perifornical region associated w/ nutritional homeostasis, blood pressure, ther

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mal regulation, neural
control of endocrine secretion and arousal - these activites might be affected b
y H
-perifornical hypothalamus contains neurons that express melanin-concentrating h
ormone (MCH), a peptide
involved in feeding behaviour
-MCH and H neurons are distinct but spatially intermingled
-one-to-one correspondence btwn neurons in lateral hypothalamus that express the
opioid receptor agonist
dynorphin and the H neurons
-H neurons receive direct projections from neurons in SCN which generates circad
ian rhythm
-antisera against H fragments show a few thousand immunoreactive neurons btwn fo
rnix and mammillothalamic
tracts, and projections from these cells to neurons in perifornical and posterio
r hypothalamus
-further projections w/ terminal fields w/i septal nuclei in basal forebrain, pr
eoptic area, paraventricular
nucleus of thalamus, central grey, locus coeruleus, pedunculopontine tegmental n
ucleus (PPT) and spinal cord
-set of projections is consistent w/ patterns of expression of 2 H GPCRs, so the
re are no other receptors for
these peptides - projection fields in humans similar to those in rodents
H are neuroexcitatory
-H immunoreactivity is associated w/ dense-core vesicles (can be seen traversing
Golgi network, along
myelinated axons and at presynaptic terminals apposed to dendritic shafts)
-accumulation of H w/i dense-core vesicles at axons terminals = H might have int
ercellular signalling activity
-bath application of synthetic Hcrt2 to mature hypothalamic neurons = inc in fre
q of postsynaptic currents
-# responsive neurons in diff brain regions consistent w/ projection & receptor
33% of hypothalamic neurons respond to Hcrt2 compared w/ 5% cerebral cortex neur
ons and 15% spinal cord
neurons - 80% of neurons w/i hypothalamic paraventricular nucleus are excited by
Hcrt2, similar results
obtained by applying Hcrt1 to locus coeruleus slices
-Hcrt2 has potent effect at presynaptic & postsynaptic receptors; in presence of
TTX, it inc the freq, but not
the amplitude of mini postsynaptic currents (presynaptic effect) and evokes an i
nc in cytoplasmic Ca by
opening plama-membrane Ca channels in arcuate postsynaptic neurons (postsynaptic
-most synaptic activity in hypothalamic circuits due to axonal release of GABA (
gamma-aminobutyric acid) or
-H, acting at axon terminals can inc release of each of these NTs (shown by whol
e cell patch clamp recording)
-Hcrt1&2 evoke rises in Ca in ~ 1/3 of hypothalamic neurons, by opening a Ca cha
-responses to H are completely blocked by the PKC-specific inhibitor bisindolylm
aleide and by phospholipase C
inhibitors, indicating that H operate through a family of GTP-binding proteins (
Gq) that activate PKC and
mobilize intracellular Ca
-Gq-activated signalling cascades result in phosphorylation of Ca channels, whic
h can inc Ca conductance and
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