Control of Gonadotropin Releasing Control of Gonadotropin Releasing Hormone (GnRH) by the Hormone (GnRH) by the

hypothalamus.hypothalamus. Release is pulsatile in all mammals looked at Release is pulsatile in all mammals looked at

(probably true for all vertebrates). (probably true for all vertebrates).

Disruption of pulsatile secretion is associated Disruption of pulsatile secretion is associated with reproductive disorders in humans.with reproductive disorders in humans.

In humans there is only one form of GnRH. In humans there is only one form of GnRH. Often referred to as LHRH due to sequence Often referred to as LHRH due to sequence homology with one of the two GnRHs found homology with one of the two GnRHs found in other vertebrates.in other vertebrates.

In Rhesus monkeys In Rhesus monkeys

There are about 2000 neurons in There are about 2000 neurons in hypothalamus that contain LHRH. hypothalamus that contain LHRH.

Early studies have suggested that Early studies have suggested that the pulse generator is physically the pulse generator is physically located in the medial-basal located in the medial-basal hypothalamus.hypothalamus.

Critical experiment- Critical experiment- complete deafferentation of that region complete deafferentation of that region

does not block pulses.does not block pulses.

Cells taken from this region and put into Cells taken from this region and put into cell culture show pulsatile release of cell culture show pulsatile release of GnRH. GnRH.

Pulses are also seen in cultured basal Pulses are also seen in cultured basal hypothalamus both rat and guinea pig. hypothalamus both rat and guinea pig.

Electrophysiological evidence also Electrophysiological evidence also supports the idea that the pulse generator supports the idea that the pulse generator is from this region. is from this region.

Volleys of action potentials (extracellular Volleys of action potentials (extracellular electrodes) have been recorded in this region electrodes) have been recorded in this region and these volleys coincide with the pulses of and these volleys coincide with the pulses of GnRH.GnRH.

These volleys have only been observed in These volleys have only been observed in the basal hypothalamus and nowhere else, the basal hypothalamus and nowhere else, suggesting that the pulses do not originate suggesting that the pulses do not originate from outside the basal hypothalamus.from outside the basal hypothalamus.

Pulsatile LHRH release is seen in tissue Pulsatile LHRH release is seen in tissue fragments of median eminence from the fragments of median eminence from the rat. rat.

This region doesn’t have the cell bodies, This region doesn’t have the cell bodies, suggesting that the pulses are initiated in the suggesting that the pulses are initiated in the axons of the LHRH neurons, not up at the cell axons of the LHRH neurons, not up at the cell bodies.bodies.

This suggests that other signals must This suggests that other signals must influence LHRH release.influence LHRH release.

One candidate in Neuropeptide Y (NPY).One candidate in Neuropeptide Y (NPY).

NPY has been shown to stimulate LHRH NPY has been shown to stimulate LHRH release from median eminence fragments release from median eminence fragments in culture.in culture.

NPY is also released in a pulsatile fashion NPY is also released in a pulsatile fashion in the stalk median eminence of the rhesus in the stalk median eminence of the rhesus monkey and these pulses appear to be monkey and these pulses appear to be synchronous with LHRH pulses.synchronous with LHRH pulses.

Note, this is not proof that the NPY is causing Note, this is not proof that the NPY is causing LHRH release.LHRH release.

There are other possible candidates.There are other possible candidates.

γ aminobutyric acid (GABA), γ aminobutyric acid (GABA), dopamine and Nitric Oxide (NO) are dopamine and Nitric Oxide (NO) are also found in the same area and all also found in the same area and all seem to be released in pulses.seem to be released in pulses.

Focus on the endogenous pulse Focus on the endogenous pulse generator.generator.

LHRH neurons appear to have intrinsic LHRH neurons appear to have intrinsic pacemaker activitypacemaker activity

Some studies have been done on the GT-1 Some studies have been done on the GT-1 cell line, a mouse line that expresses the cell line, a mouse line that expresses the mouse GnRH transcript.mouse GnRH transcript.

GT-1 cells release LHRH in pulses with GT-1 cells release LHRH in pulses with intervals of about 22-30 minutes.intervals of about 22-30 minutes.

These intervals are similar to the intervals These intervals are similar to the intervals seen in the mouse seen in the mouse in vivoin vivo..

This interval is not the same as seen This interval is not the same as seen in primates.in primates.

Primate LHRH neurons in culture Primate LHRH neurons in culture also release LHRH in pulses.also release LHRH in pulses.

Interval of around 43-44 minutes.Interval of around 43-44 minutes.

LHRH neurons from 2 sources in LHRH neurons from 2 sources in rhesus brain had same interval.rhesus brain had same interval.

This suggests that there is an endogenous This suggests that there is an endogenous frequency.frequency.

Frequency in cultured cells is approximately Frequency in cultured cells is approximately the same as in adult animals the same as in adult animals in vivoin vivo..

In cultured LHRH neurons, In cultured LHRH neurons, electrophysiological studies have electrophysiological studies have demonstrated that they show oscillatory demonstrated that they show oscillatory bursting activity (bursting activity (i.e.i.e. trains of action trains of action potential).potential).

However, it is not know for certain if the APs However, it is not know for certain if the APs are directly associated with LHRH release.are directly associated with LHRH release.

In addition to the electrical activity, In addition to the electrical activity, cultured LHRH neurons show oscillatory cultured LHRH neurons show oscillatory Ca2+ activity.Ca2+ activity.

When cultured individually, these When cultured individually, these neurons all have individual frequencies.neurons all have individual frequencies.

However, when cultured together they However, when cultured together they synchronize their Ca2+ oscillations.synchronize their Ca2+ oscillations.

The synchronous activity has a frequency The synchronous activity has a frequency similar to the similar to the in vivoin vivo frequency. frequency.

A similar system of activity has been A similar system of activity has been shown to correlate to insulin release in shown to correlate to insulin release in β cells.β cells.

However, a link still needs to be However, a link still needs to be established in LHRH neurons.established in LHRH neurons.

Mechanism of communication between Mechanism of communication between LHRH neurons:LHRH neurons:

Possibilities:Possibilities: Some sort of synaptic transmission?Some sort of synaptic transmission? Electrical coupling (Electrical coupling (i.e.i.e. gap junctions)? gap junctions)? Some diffusible messenger (Some diffusible messenger (i.e.i.e. paracrine paracrine

signaling)?signaling)?

GT-1 cells have been demonstrated to GT-1 cells have been demonstrated to form both synapses and gap junctions in form both synapses and gap junctions in culture.culture.

However, it has been shown that LHRH However, it has been shown that LHRH neurons grown on separate coverslips neurons grown on separate coverslips (no physical contact) also synchronize.(no physical contact) also synchronize.

Thus, we can rule out synaptic Thus, we can rule out synaptic transmission and gap junctions as transmission and gap junctions as potential methods of cell communication.potential methods of cell communication.

This leaves paracrine signaling.This leaves paracrine signaling.

NO synthase mRNA has been found NO synthase mRNA has been found in GT-1 cells.in GT-1 cells.

NO would be an ideal candidate.NO would be an ideal candidate. Small molecule.Small molecule. Diffuses rapidly.Diffuses rapidly. Highly soluble.Highly soluble. Penetrates membranes easily.Penetrates membranes easily.

In LHRH neurons:In LHRH neurons:

Show characteristics of Show characteristics of neuroendocrine cells.neuroendocrine cells.

Depolarization causes release of Depolarization causes release of LHRH (induced depolarization).LHRH (induced depolarization).

Depolarization also causes Ca2+ Depolarization also causes Ca2+ oscillations.oscillations.

In GT-1 cells:In GT-1 cells: Depolarization by high extracellular K+ Depolarization by high extracellular K+

causes LHRH release.causes LHRH release.

Treatment with Veratridine (VG Na+ Treatment with Veratridine (VG Na+ channel opener) causes LHRH release.channel opener) causes LHRH release.

Both these treatments also cause LHRH Both these treatments also cause LHRH release from cultured fetal rhesus monkey release from cultured fetal rhesus monkey LHRH neurons.LHRH neurons.

Na+ channel blockers (Na+ channel blockers (i.e.i.e. tetrodotoxin) tetrodotoxin) will block LHRH release.will block LHRH release.

LHRH release is dependent on Ca2+ LHRH release is dependent on Ca2+ influx.influx.

Suggests the possibility that Ca2+ influx Suggests the possibility that Ca2+ influx may be involved in the pulse generation may be involved in the pulse generation (rather than being a result of pulse (rather than being a result of pulse generation).generation).

L-type Ca2+ channels have been found in L-type Ca2+ channels have been found in GT-1 cells as well as monkey LHRH cells.GT-1 cells as well as monkey LHRH cells. This is a voltage-gated Ca2+ channel.This is a voltage-gated Ca2+ channel. Characterized by nifedipine blockade, but Characterized by nifedipine blockade, but

not blockade by ώ conotoxin. Also, they are not blockade by ώ conotoxin. Also, they are activated by Bay-K8644.activated by Bay-K8644.

Bay-K8644 does stimulate LHRH release in both cell Bay-K8644 does stimulate LHRH release in both cell systems.systems.

   L-type channels have been shown to be involved in Ca2+-L-type channels have been shown to be involved in Ca2+-

activated Ca2+ influx.activated Ca2+ influx.   Ca2+-activated Ca2+ influx has been identified in GT-1 Ca2+-activated Ca2+ influx has been identified in GT-1

cells.cells.   Treatment with thapsigargan or cyanide-p-Treatment with thapsigargan or cyanide-p-

trifluoromethoxy-phenyl-hydrazone (FCCP; induces Ca2+ trifluoromethoxy-phenyl-hydrazone (FCCP; induces Ca2+ release from mitochondria) increased [Ca2+]i and release from mitochondria) increased [Ca2+]i and induced oscillations.induced oscillations.

Similar treatments (Similar treatments (i.e.i.e. thapsigargan or ryanodine) thapsigargan or ryanodine) induced calcium oscillations in fetal monkey LHRH induced calcium oscillations in fetal monkey LHRH neurons.neurons.

Other potential influences:Other potential influences:

Neuropeptide Y (NPY)Neuropeptide Y (NPY) 36 aa peptide36 aa peptide

These studies were done These studies were done in vivoin vivo on on monkeys, using the push-pull monkeys, using the push-pull cannula technique.cannula technique.

PerfusateIn

Under pressure

Effluent out

Perfusate percolates Through interstitial space

Skull

Double-barreledcannula

Push-pull cannulaPush-pull cannula

Infusion (8 hour) of antisense Infusion (8 hour) of antisense oligonucleotide against NPY blocks both oligonucleotide against NPY blocks both the NPY pulses and LHRH pulses.the NPY pulses and LHRH pulses.

Infusion of anti-NPY antisera will also Infusion of anti-NPY antisera will also block both LHRH and NPY pulses.block both LHRH and NPY pulses.

Thus, good evidence that NPY is playing Thus, good evidence that NPY is playing a role in at least regulating the LHRH a role in at least regulating the LHRH pulses, but is it the only factor? pulses, but is it the only factor?

Norepinephrine (NE) may also be involved.Norepinephrine (NE) may also be involved.

There are NE containing neurons in the There are NE containing neurons in the same region of the stalk median eminence.same region of the stalk median eminence.

Adrenergic input has been shown to Adrenergic input has been shown to modulate LH and LHRH release.modulate LH and LHRH release.

LH pulses (from pituitary) and intermittent LH pulses (from pituitary) and intermittent bursts of multiple unit activity (in ME) occur in bursts of multiple unit activity (in ME) occur in close association with LHRH pulses and can be close association with LHRH pulses and can be suppressed with α-adrenergic blockers suppressed with α-adrenergic blockers phentolamine and prazosin (α1-specific).phentolamine and prazosin (α1-specific).

Push-pull cannula results show that Push-pull cannula results show that release of NE from stalk-ME is pulsatile release of NE from stalk-ME is pulsatile and the pulses are synchronous with and the pulses are synchronous with LHRH pulses.LHRH pulses.

Direct infusion of NE, or α-adrenergic Direct infusion of NE, or α-adrenergic agonists, into stalk-ME stimulates release agonists, into stalk-ME stimulates release of LHRH.of LHRH.

The α1-blocker prazosin reduces pulse The α1-blocker prazosin reduces pulse amplitude, but not pulse frequency.amplitude, but not pulse frequency.

β- and α2- antagonists have no effect.β- and α2- antagonists have no effect.

Infusion of NE into stalk-ME also Infusion of NE into stalk-ME also stimulates the release of prostaglandin stimulates the release of prostaglandin E2 (PGE2).E2 (PGE2).

PGE2 infusion will also stimulate LHRH PGE2 infusion will also stimulate LHRH release.release.

Thus, NE may work directly, or through Thus, NE may work directly, or through PGE2 , or both.PGE2 , or both.

Organ culture on ME fragments has Organ culture on ME fragments has shown that NE effects are mediated shown that NE effects are mediated through PGE2 . through PGE2 .

Interactions between NPY and NE/ Interactions between NPY and NE/ PGE2 PGE2

NPY infused into stalk-ME of NPY infused into stalk-ME of prazosin-treated monkeys stimulated prazosin-treated monkeys stimulated LHRH release.LHRH release.

This suggests that NPY is working This suggests that NPY is working independently of NE/ PGE2.independently of NE/ PGE2.

Also, α1- adrenergic stimulation with Also, α1- adrenergic stimulation with methoxyamine in monkeys treated with methoxyamine in monkeys treated with antisense oligonucleotide for NPY still antisense oligonucleotide for NPY still had LHRH release suggesting that NE had LHRH release suggesting that NE effects are not working through NPY effects are not working through NPY neurons. neurons.

Thus, it appears that NPY and NE are Thus, it appears that NPY and NE are working independently to modulate working independently to modulate LHRH release.LHRH release.

γ-aminobutyric acid γ-aminobutyric acid (GABA)(GABA)

This is the major inhibitory This is the major inhibitory neurotransmitter in the CNS and it is neurotransmitter in the CNS and it is found in the hypothalamus.found in the hypothalamus.

Early studies, involving infusion of Early studies, involving infusion of GABA into the CSF in the third GABA into the CSF in the third ventricle, stimulated LHRH releaseventricle, stimulated LHRH release

This was also observed in explants of This was also observed in explants of hypothalamic tissue kept in culture.hypothalamic tissue kept in culture.

However, other studies were contradictory.However, other studies were contradictory.

A more recent observation is that GABA may A more recent observation is that GABA may change in activity with changing age or change in activity with changing age or developmental status.developmental status.

In rats, GABA appears to stimulate LHRH In rats, GABA appears to stimulate LHRH release in juveniles, but becomes inhibitory at release in juveniles, but becomes inhibitory at puberty.puberty.

More recently, studies of GT-1 cells and LHRH More recently, studies of GT-1 cells and LHRH neurons (from mouse olfactory placode) show neurons (from mouse olfactory placode) show that GABA actually stimulates LHRH release, that GABA actually stimulates LHRH release, as well as increasing intracellular Ca2+ as well as increasing intracellular Ca2+ oscillations and membrane potentials oscillations and membrane potentials (depolarization).(depolarization).

In monkeys (using the push-pull In monkeys (using the push-pull cannula)…cannula)…

GABA tonically inhibits LHRH GABA tonically inhibits LHRH release before puberty.release before puberty.

GABA levels in ME are much higher GABA levels in ME are much higher in pre-pubertal animals and levels in pre-pubertal animals and levels drop by mid-puberty drop by mid-puberty

Another neurotransmitter found in the ME Another neurotransmitter found in the ME region is glutamate.region is glutamate.

It is an agonist of the excitatory amino It is an agonist of the excitatory amino acid system, working on NMDA receptors.acid system, working on NMDA receptors.

It has also been shown to stimulate LHRH It has also been shown to stimulate LHRH release.release.

Stimulation of Stimulation of NN-methyl-D-aspartate -methyl-D-aspartate (NMDA) receptors can induce precocious (NMDA) receptors can induce precocious puberty in rats.puberty in rats.

There are potential interactions between There are potential interactions between GABA and glutamate.GABA and glutamate.

GABA is synthesized from glutamate.GABA is synthesized from glutamate.

This raises the possibility that GABA and This raises the possibility that GABA and glutamate levels may be related.glutamate levels may be related.

Infusion of antisense oligonucleotides for Infusion of antisense oligonucleotides for enzymes involved in the synthesis of GABA enzymes involved in the synthesis of GABA from glutamate cause an increase in LHRH from glutamate cause an increase in LHRH in pre-pubertal monkeys.in pre-pubertal monkeys.

i.ei.e. when GABA synthesis was inhibited, . when GABA synthesis was inhibited, LHRH levels rose.LHRH levels rose.

Was this due to a local increase of Was this due to a local increase of glutamate, once GABA synthesis was glutamate, once GABA synthesis was stopped?stopped?

   Or, did reduction of GABA simply Or, did reduction of GABA simply

unmask an ongoing glutamate unmask an ongoing glutamate stimulation?stimulation?

Several studies suggest the former.Several studies suggest the former.

In pre-pubertal monkeys, the glutamate In pre-pubertal monkeys, the glutamate levels in the stalk ME are very low. levels in the stalk ME are very low.

When GABA synthesis is blocked, these When GABA synthesis is blocked, these levels rise.levels rise.

However, there are studies that However, there are studies that suggest the former may also be suggest the former may also be occurring. occurring.

The probability is that both occur to The probability is that both occur to some extent (and there may be some extent (and there may be variability between species).variability between species).

As mentioned before, NO may be playing a role As mentioned before, NO may be playing a role and is an ideal candidate.and is an ideal candidate.

Pulsatile LHRH release occurs even when the cell bodies Pulsatile LHRH release occurs even when the cell bodies are not present.are not present.

This suggests some form of pre-synaptic stimulation at This suggests some form of pre-synaptic stimulation at the neuroterminals of the LHRH neurons.the neuroterminals of the LHRH neurons.

Histological studies have failed to show the presence of Histological studies have failed to show the presence of pre-synaptic synapses in the right area of the pre-synaptic synapses in the right area of the hypothalamus.hypothalamus.

Similar results are seen in cultures cells. Cells that are Similar results are seen in cultures cells. Cells that are physically separated, yet cultured in the same dish, show physically separated, yet cultured in the same dish, show synchrony of LHRH pulses and Ca2+ oscillations.synchrony of LHRH pulses and Ca2+ oscillations.

This is strong evidence that there is a chemical This is strong evidence that there is a chemical mediator (i.e.some kind of paracrine signaling), mediator (i.e.some kind of paracrine signaling), which coordinates the LHRH release.which coordinates the LHRH release.

In push-pull cannula experiments, infusion of In push-pull cannula experiments, infusion of the NO precursor L-argenine stimulated both the NO precursor L-argenine stimulated both NPY and LHRH.NPY and LHRH.

Infusion of D-argenine had no effect (the Infusion of D-argenine had no effect (the dextrorotary form cannot be converted into dextrorotary form cannot be converted into NO).NO).

   The enzymes involved in NO synthesis are The enzymes involved in NO synthesis are

present in an adjacent area of the present in an adjacent area of the hypothalamus.hypothalamus.

   This means that NO is available in the area in This means that NO is available in the area in

question.question.

Finally, glial cells may also be playing Finally, glial cells may also be playing a role. a role.

In other systems, glial cells have been In other systems, glial cells have been shown to modify, or affect the release shown to modify, or affect the release of neurotransmitters and of neurotransmitters and neurohormones.neurohormones.

In this system, circumstantial In this system, circumstantial evidence suggests this possibility.evidence suggests this possibility.

The endogenous pacemaker seems to The endogenous pacemaker seems to be located in, or near the be located in, or near the neuroterminals of the LHRH cells (and neuroterminals of the LHRH cells (and not near the cells bodies).not near the cells bodies).

No pre-synaptic synaptic connections No pre-synaptic synaptic connections have been identified in the area.have been identified in the area.

Glial cells Glial cells AREARE present around the present around the neuro-terminals.neuro-terminals.

It is known that glial cells play an important It is known that glial cells play an important role in regulating release of the hormones of role in regulating release of the hormones of the the pars nervosa,pars nervosa, an analogous system to that an analogous system to that of the stalk ME.of the stalk ME.

   The proposed mechanism for this interaction The proposed mechanism for this interaction

is that glial cells may release the kallekrein is that glial cells may release the kallekrein bradykinin. bradykinin.

   Bradykinin, in turn, would stimulate glutamate Bradykinin, in turn, would stimulate glutamate

release from astrocytes located around the release from astrocytes located around the neuroterminals.neuroterminals.

   This would have an effect on LHRH release This would have an effect on LHRH release

from the neuroterminals themselves.from the neuroterminals themselves.

LHRHGABA

Glu

GABA

GABA

Glu

NPYNE

NO

GliaGlia

Portal blood

Master Pacemaker?