Transcript Biol 155 Human Physiology - University of British Columbia

Control of Gonadotropin Releasing Hormone
(GnRH) by the hypothalamus.
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Release is pulsatile in all mammals looked at (probably
true for all vertebrates).
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Disruption of pulsatile secretion is associated with
reproductive disorders in humans.
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In humans there is only one form of GnRH. Often
referred to as LHRH due to sequence homology with
one of the two GnRHs found in other vertebrates.
In Rhesus monkeys
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There are about 2000 neurons in hypothalamus
that contain LHRH.
Early studies have suggested that the pulse
generator is physically located in the medialbasal hypothalamus.
Critical experiment
complete deafferentation of that region does not
block pulses.
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Cells taken from this region and put into cell
culture show pulsatile release of GnRH.
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Pulses are also seen in cultured basal
hypothalamus both rat and guinea pig.
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Electrophysiological evidence also supports the
idea that the pulse generator is from this region.
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Volleys of action potentials (extracellular electrodes)
have been recorded in this region and these volleys
coincide with the pulses of GnRH.
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These volleys have only been observed in the basal
hypothalamus and nowhere else, suggesting that the
pulses do not originate from outside the basal
hypothalamus.
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Pulsatile LHRH release is seen in tissue fragments of
median eminence from the rat.
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This region doesn’t have the cell bodies, suggesting that the
pulses are initiated in the axons of the LHRH neurons, not
up at the cell bodies.
This suggests that other signals must influence LHRH
release.
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One candidate in Neuropeptide Y (NPY).
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NPY has been shown to stimulate LHRH release from
median eminence fragments in culture.
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NPY is also released in a pulsatile fashion in the stalk
median eminence of the rhesus monkey and these
pulses appear to be synchronous with LHRH pulses.
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Note, this is not proof that the NPY is causing LHRH
release.
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There are other possible candidates.
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γ aminobutyric acid (GABA), dopamine and
Nitric Oxide (NO) are also found in the same
area and all seem to be released in pulses.
Focus on the endogenous pulse generator.
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LHRH neurons appear to have intrinsic pacemaker
activity
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Some studies have been done on the GT-1 cell line, a
mouse line that expresses the mouse GnRH transcript.
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GT-1 cells release LHRH in pulses with intervals of
about 22-30 minutes.
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These intervals are similar to the intervals seen in the
mouse in vivo.
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This interval is not the same as seen in primates.
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Primate LHRH neurons in culture also release
LHRH in pulses.
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Interval of around 43-44 minutes.
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LHRH neurons from 2 sources in rhesus brain
had same interval.
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This suggests that there is an endogenous frequency.
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Frequency in cultured cells is approximately the same as
in adult animals in vivo.
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In cultured LHRH neurons, electrophysiological studies
have demonstrated that they show oscillatory bursting
activity (i.e. trains of action potential).
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However, it is not know for certain if the APs are
directly associated with LHRH release.
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In addition to the electrical activity, cultured
LHRH neurons show oscillatory Ca2+ activity.
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When cultured individually, these neurons all
have individual frequencies.
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However, when cultured together they
synchronize their Ca2+ oscillations.
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The synchronous activity has a frequency similar
to the in vivo frequency.
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A similar system of activity has been shown to
correlate to insulin release in β cells.
However, a link still needs to be established in
LHRH neurons.
Mechanism of communication between LHRH
neurons:
Possibilities:
Some sort of synaptic transmission?
 Electrical coupling (i.e. gap junctions)?
 Some diffusible messenger (i.e. paracrine signaling)?
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GT-1 cells have been demonstrated to form
both synapses and gap junctions in culture.
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However, it has been shown that LHRH
neurons grown on separate coverslips (no
physical contact) also synchronize.
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Thus, we can rule out synaptic transmission and
gap junctions as potential methods of cell
communication.
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This leaves paracrine signaling.
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NO synthase mRNA has been found in GT-1
cells.
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NO would be an ideal candidate.
Small molecule.
 Diffuses rapidly.
 Highly soluble.
 Penetrates membranes easily.
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In LHRH neurons:
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Show characteristics of neuroendocrine cells.
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Depolarization causes release of LHRH
(induced depolarization).
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Depolarization also causes Ca2+ oscillations.
In GT-1 cells:
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Depolarization by high extracellular K+ causes LHRH
release.
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Treatment with Veratridine (VG Na+ channel opener)
causes LHRH release.
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Both these treatments also cause LHRH release from
cultured fetal rhesus monkey LHRH neurons.
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Na+ channel blockers (i.e. tetrodotoxin) will block
LHRH release.
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LHRH release is dependent on Ca2+ influx.
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Suggests the possibility that Ca2+ influx may be
involved in the pulse generation (rather than
being a result of pulse generation).
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L-type Ca2+ channels have been found in GT-1
cells as well as monkey LHRH cells.
This is a voltage-gated Ca2+ channel.
 Characterized by nifedipine blockade, but not
blockade by ώ conotoxin. Also, they are activated by
Bay-K8644.
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Bay-K8644 does stimulate LHRH release in both cell systems.
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L-type channels have been shown to be involved in Ca2+activated Ca2+ influx.
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Ca2+-activated Ca2+ influx has been identified in GT-1 cells.
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Treatment with thapsigargan or cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP; induces Ca2+ release from
mitochondria) increased [Ca2+]i and induced oscillations.
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Similar treatments (i.e. thapsigargan or ryanodine) induced
calcium oscillations in fetal monkey LHRH neurons.
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Other potential influences:
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Neuropeptide Y (NPY)
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36 aa peptide
These studies were done in vivo on monkeys, using
the push-pull cannula technique.
Perfusate
In
Under pressure
Effluent out
Skull
Push-pull cannula
Doublebarreled
cannula
Perfusate percolates
Through interstitial space
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Infusion (8 hour) of antisense oligonucleotide
against NPY blocks both the NPY pulses and
LHRH pulses.
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Infusion of anti-NPY antisera will also block
both LHRH and NPY pulses.
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Thus, good evidence that NPY is playing a role
in at least regulating the LHRH pulses, but is it
the only factor?
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Norepinephrine (NE) may also be involved.
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There are NE containing neurons in the same region of
the stalk median eminence.
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Adrenergic input has been shown to modulate LH and
LHRH release.
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LH pulses (from pituitary) and intermittent bursts of
multiple unit activity (in ME) occur in close association with
LHRH pulses and can be suppressed with α-adrenergic
blockers phentolamine and prazosin (α1-specific).
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Push-pull cannula results show that release of
NE from stalk-ME is pulsatile and the pulses are
synchronous with LHRH pulses.
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Direct infusion of NE, or α-adrenergic agonists,
into stalk-ME stimulates release of LHRH.
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The α1-blocker prazosin reduces pulse
amplitude, but not pulse frequency.
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β- and α2- antagonists have no effect.
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Infusion of NE into stalk-ME also stimulates
the release of prostaglandin E2 (PGE2).
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PGE2 infusion will also stimulate LHRH
release.
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Thus, NE may work directly, or through PGE2 ,
or both.
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Organ culture on ME fragments has shown that
NE effects are mediated through PGE2 .
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Interactions between NPY and NE/ PGE2
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NPY infused into stalk-ME of prazosin-treated
monkeys stimulated LHRH release.
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This suggests that NPY is working
independently of NE/ PGE2.
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Also, α1- adrenergic stimulation with
methoxyamine in monkeys treated with
antisense oligonucleotide for NPY still had
LHRH release suggesting that NE effects are
not working through NPY neurons.
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Thus, it appears that NPY and NE are working
independently to modulate LHRH release.
γ-aminobutyric acid (GABA)
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This is the major inhibitory neurotransmitter in
the CNS and it is found in the hypothalamus.
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Early studies, involving infusion of GABA into
the CSF in the third ventricle, stimulated LHRH
release
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This was also observed in explants of
hypothalamic tissue kept in culture.
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However, other studies were contradictory.
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A more recent observation is that GABA may change
in activity with changing age or developmental status.
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In rats, GABA appears to stimulate LHRH release in
juveniles, but becomes inhibitory at puberty.
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More recently, studies of GT-1 cells and LHRH
neurons (from mouse olfactory placode) show that
GABA actually stimulates LHRH release, as well as
increasing intracellular Ca2+ oscillations and membrane
potentials (depolarization).
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In monkeys (using the push-pull cannula)…
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GABA tonically inhibits LHRH release before
puberty.
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GABA levels in ME are much higher in prepubertal animals and levels drop by mid-puberty
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Another neurotransmitter found in the ME region is
glutamate.
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It is an agonist of the excitatory amino acid system,
working on NMDA receptors.
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It has also been shown to stimulate LHRH release.
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Stimulation of N-methyl-D-aspartate (NMDA)
receptors can induce precocious puberty in rats.
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There are potential interactions between GABA and
glutamate.
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GABA is synthesized from glutamate.
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This raises the possibility that GABA and glutamate
levels may be related.
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Infusion of antisense oligonucleotides for enzymes
involved in the synthesis of GABA from glutamate
cause an increase in LHRH in pre-pubertal monkeys.
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i.e. when GABA synthesis was inhibited, LHRH levels
rose.
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Was this due to a local increase of glutamate,
once GABA synthesis was stopped?
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Or, did reduction of GABA simply unmask an
ongoing glutamate stimulation?
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Several studies suggest the former.
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In pre-pubertal monkeys, the glutamate levels in
the stalk ME are very low.
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When GABA synthesis is blocked, these levels
rise.
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However, there are studies that suggest the
former may also be occurring.
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The probability is that both occur to some
extent (and there may be variability between
species).
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As mentioned before, NO may be playing a role and is an ideal
candidate.
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Pulsatile LHRH release occurs even when the cell bodies are not present.
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This suggests some form of pre-synaptic stimulation at the
neuroterminals of the LHRH neurons.
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Histological studies have failed to show the presence of pre-synaptic
synapses in the right area of the hypothalamus.
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Similar results are seen in cultures cells. Cells that are physically
separated, yet cultured in the same dish, show synchrony of LHRH
pulses and Ca2+ oscillations.
This is strong evidence that there is a chemical mediator
(i.e.some kind of paracrine signaling), which coordinates the
LHRH release.
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In push-pull cannula experiments, infusion of the NO
precursor L-argenine stimulated both NPY and LHRH.
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Infusion of D-argenine had no effect (the dextrorotary
form cannot be converted into NO).
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The enzymes involved in NO synthesis are present in
an adjacent area of the hypothalamus.
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This means that NO is available in the area in question.
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Finally, glial cells may also be playing a role.
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In other systems, glial cells have been shown to
modify, or affect the release of neurotransmitters
and neurohormones.
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In this system, circumstantial evidence suggests
this possibility.
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The endogenous pacemaker seems to be located in,
or near the neuroterminals of the LHRH cells (and
not near the cells bodies).
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No pre-synaptic synaptic connections have been
identified in the area.
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Glial cells ARE present around the neuroterminals.
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It is known that glial cells play an important role in
regulating release of the hormones of the pars nervosa,
an analogous system to that of the stalk ME.
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The proposed mechanism for this interaction is that
glial cells may release the kallekrein bradykinin.
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Bradykinin, in turn, would stimulate glutamate release
from astrocytes located around the neuroterminals.
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This would have an effect on LHRH release from the
neuroterminals themselves.
NPY
NE
LHRH
GABA
GABA
Glu
Glu
NO
Master
Pacemaker?
GABA
Glia
Glia
Portal blood