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Control of Gonadotropin Releasing Hormone (GnRH) by the hypothalamus. Release is pulsatile in all mammals looked at (probably true for all vertebrates).

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Presentation on theme: "Control of Gonadotropin Releasing Hormone (GnRH) by the hypothalamus. Release is pulsatile in all mammals looked at (probably true for all vertebrates)."— Presentation transcript:

1 Control of Gonadotropin Releasing Hormone (GnRH) by the hypothalamus. Release is pulsatile in all mammals looked at (probably true for all vertebrates). Release is pulsatile in all mammals looked at (probably true for all vertebrates). Disruption of pulsatile secretion is associated with reproductive disorders in humans. Disruption of pulsatile secretion is associated with reproductive disorders in humans. 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 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.

2 In Rhesus monkeys There are about 2000 neurons in hypothalamus that contain LHRH. There are about 2000 neurons in hypothalamus that contain LHRH. Early studies have suggested that the pulse generator is physically located in the medial- basal hypothalamus. Early studies have suggested that the pulse generator is physically located in the medial- basal hypothalamus. Critical experiment- Critical experiment- complete deafferentation of that region does not block pulses. complete deafferentation of that region does not block pulses.

3 Cells taken from this region and put into cell culture show pulsatile release of GnRH. Cells taken from this region and put into cell culture show pulsatile release of GnRH. Pulses are also seen in cultured basal hypothalamus both rat and guinea pig. Pulses are also seen in cultured basal hypothalamus both rat and guinea pig. Electrophysiological evidence also supports the idea that the pulse generator is from this region. Electrophysiological evidence also supports the idea that the pulse generator is from this region. Volleys of action potentials (extracellular electrodes) have been recorded in this region and these volleys coincide with the pulses of GnRH. Volleys of action potentials (extracellular electrodes) have been recorded in this region and these volleys coincide with the pulses of GnRH.

4 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. 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. Pulsatile LHRH release is seen in tissue fragments of median eminence from the rat. Pulsatile LHRH release is seen in tissue fragments of median eminence from the rat. 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 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. This suggests that other signals must influence LHRH release.

5 One candidate in Neuropeptide Y (NPY). One candidate in Neuropeptide Y (NPY). NPY has been shown to stimulate LHRH release from median eminence fragments in culture. NPY has been shown to stimulate LHRH release from median eminence fragments in culture. 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. 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. Note, this is not proof that the NPY is causing LHRH release. Note, this is not proof that the NPY is causing LHRH release.

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

7 Focus on the endogenous pulse generator. LHRH neurons appear to have intrinsic pacemaker activity LHRH neurons appear to have intrinsic pacemaker activity Some studies have been done on the GT-1 cell line, a mouse line that expresses the mouse GnRH transcript. Some studies have been done on the GT-1 cell line, a mouse line that expresses the mouse GnRH transcript. GT-1 cells release LHRH in pulses with intervals of about minutes. GT-1 cells release LHRH in pulses with intervals of about minutes. These intervals are similar to the intervals seen in the mouse in vivo. These intervals are similar to the intervals seen in the mouse in vivo.

8 This interval is not the same as seen in primates. This interval is not the same as seen in primates. Primate LHRH neurons in culture also release LHRH in pulses. Primate LHRH neurons in culture also release LHRH in pulses. Interval of around minutes. Interval of around minutes. LHRH neurons from 2 sources in rhesus brain had same interval. LHRH neurons from 2 sources in rhesus brain had same interval.

9 This suggests that there is an endogenous frequency. This suggests that there is an endogenous frequency. Frequency in cultured cells is approximately the same as in adult animals in vivo. Frequency in cultured cells is approximately the same as in adult animals in vivo. In cultured LHRH neurons, electrophysiological studies have demonstrated that they show oscillatory bursting activity (i.e. trains of action potential). In cultured LHRH neurons, electrophysiological studies have demonstrated that they show oscillatory bursting activity (i.e. trains of action potential). However, it is not know for certain if the APs are directly associated with LHRH release. However, it is not know for certain if the APs are directly associated with LHRH release.

10 In addition to the electrical activity, cultured LHRH neurons show oscillatory Ca2+ activity. In addition to the electrical activity, cultured LHRH neurons show oscillatory Ca2+ activity. When cultured individually, these neurons all have individual frequencies. When cultured individually, these neurons all have individual frequencies. However, when cultured together they synchronize their Ca2+ oscillations. However, when cultured together they synchronize their Ca2+ oscillations. The synchronous activity has a frequency similar to the in vivo frequency. The synchronous activity has a frequency similar to the in vivo frequency.

11 A similar system of activity has been shown to correlate to insulin release in β cells. 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. However, a link still needs to be established in LHRH neurons. Mechanism of communication between LHRH neurons: Mechanism of communication between LHRH neurons: Possibilities: Possibilities: Some sort of synaptic transmission? Some sort of synaptic transmission? Electrical coupling (i.e. gap junctions)? Electrical coupling (i.e. gap junctions)? Some diffusible messenger (i.e. paracrine signaling)? Some diffusible messenger (i.e. paracrine signaling)?

12 GT-1 cells have been demonstrated to form both synapses and gap junctions in culture. GT-1 cells have been demonstrated to form both synapses and gap junctions in culture. However, it has been shown that LHRH neurons grown on separate coverslips (no physical contact) also synchronize. However, it has been shown that LHRH neurons grown on separate coverslips (no physical contact) also synchronize. Thus, we can rule out synaptic transmission and gap junctions as potential methods of cell communication. Thus, we can rule out synaptic transmission and gap junctions as potential methods of cell communication.

13 This leaves paracrine signaling. This leaves paracrine signaling. NO synthase mRNA has been found in GT-1 cells. NO synthase mRNA has been found 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.

14 In LHRH neurons: Show characteristics of neuroendocrine cells. Show characteristics of neuroendocrine cells. Depolarization causes release of LHRH (induced depolarization). Depolarization causes release of LHRH (induced depolarization). Depolarization also causes Ca2+ oscillations. Depolarization also causes Ca2+ oscillations.

15 In GT-1 cells: Depolarization by high extracellular K+ causes LHRH release. Depolarization by high extracellular K+ causes LHRH release. Treatment with Veratridine (VG Na+ channel opener) causes LHRH release. Treatment with Veratridine (VG Na+ channel opener) causes LHRH release. Both these treatments also cause LHRH release from cultured fetal rhesus monkey LHRH neurons. Both these treatments also cause LHRH release from cultured fetal rhesus monkey LHRH neurons. Na+ channel blockers (i.e. tetrodotoxin) will block LHRH release. Na+ channel blockers (i.e. tetrodotoxin) will block LHRH release.

16 LHRH release is dependent on Ca2+ influx. LHRH release is dependent on Ca2+ influx. Suggests the possibility that Ca2+ influx may be involved in the pulse generation (rather than being a result of pulse generation). Suggests the possibility that Ca2+ influx may be involved in the pulse generation (rather than being a result of pulse generation). L-type Ca2+ channels have been found in GT-1 cells as well as monkey LHRH cells. L-type Ca2+ channels have been found in 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 not blockade by ώ conotoxin. Also, they are activated by Bay-K8644. Characterized by nifedipine blockade, but not blockade by ώ conotoxin. Also, they are activated by Bay-K8644.

17 Bay-K8644 does stimulate LHRH release in both cell systems. Bay-K8644 does stimulate LHRH release in both cell systems. L-type channels have been shown to be involved in Ca2+- activated Ca2+ influx. L-type channels have been shown to be involved in Ca2+- activated Ca2+ influx. Ca2+-activated Ca2+ influx has been identified in GT-1 cells. Ca2+-activated Ca2+ influx has been identified in GT-1 cells. Treatment with thapsigargan or cyanide-p-trifluoromethoxy- phenyl-hydrazone (FCCP; induces Ca2+ release from mitochondria) increased [Ca2+]i and induced oscillations. Treatment with thapsigargan or cyanide-p-trifluoromethoxy- phenyl-hydrazone (FCCP; induces Ca2+ release from mitochondria) increased [Ca2+]i and induced oscillations. Similar treatments (i.e. thapsigargan or ryanodine) induced calcium oscillations in fetal monkey LHRH neurons. Similar treatments (i.e. thapsigargan or ryanodine) induced calcium oscillations in fetal monkey LHRH neurons.

18 Other potential influences: Other potential influences: Neuropeptide Y (NPY) Neuropeptide Y (NPY) 36 aa peptide 36 aa peptide These studies were done in vivo on monkeys, using the push-pull cannula technique.

19 Perfusate In Under pressure Effluent out Perfusate percolates Through interstitial space Skull Double- barreled cannula Push-pull cannula

20 Infusion (8 hour) of antisense oligonucleotide against NPY blocks both the NPY pulses and LHRH pulses. Infusion (8 hour) of antisense oligonucleotide against NPY blocks both the NPY pulses and LHRH pulses. Infusion of anti-NPY antisera will also block both LHRH and NPY pulses. Infusion of anti-NPY antisera will also block both LHRH and NPY pulses. Thus, good evidence that NPY is playing a role in at least regulating the LHRH pulses, but is it the only factor? Thus, good evidence that NPY is playing a role in at least regulating the LHRH pulses, but is it the only factor?

21 Norepinephrine (NE) may also be involved. Norepinephrine (NE) may also be involved. There are NE containing neurons in the same region of the stalk median eminence. There are NE containing neurons in the same region of the stalk median eminence. Adrenergic input has been shown to modulate LH and LHRH release. Adrenergic input has been shown to modulate LH and LHRH release. 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). 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).

22 Push-pull cannula results show that release of NE from stalk-ME is pulsatile and the pulses are synchronous with LHRH pulses. Push-pull cannula results show that release of NE from stalk-ME is pulsatile and the pulses are synchronous with LHRH pulses. Direct infusion of NE, or α-adrenergic agonists, into stalk-ME stimulates release of LHRH. Direct infusion of NE, or α-adrenergic agonists, into stalk-ME stimulates release of LHRH. The α1-blocker prazosin reduces pulse amplitude, but not pulse frequency. The α1-blocker prazosin reduces pulse amplitude, but not pulse frequency. β- and α2- antagonists have no effect. β- and α2- antagonists have no effect.

23 Infusion of NE into stalk-ME also stimulates the release of prostaglandin E2 (PGE2). Infusion of NE into stalk-ME also stimulates the release of prostaglandin E2 (PGE2). PGE2 infusion will also stimulate LHRH release. PGE2 infusion will also stimulate LHRH release. Thus, NE may work directly, or through PGE2, or both. Thus, NE may work directly, or through PGE2, or both. Organ culture on ME fragments has shown that NE effects are mediated through PGE2. Organ culture on ME fragments has shown that NE effects are mediated through PGE2.

24 Interactions between NPY and NE/ PGE2 Interactions between NPY and NE/ PGE2 NPY infused into stalk-ME of prazosin-treated monkeys stimulated LHRH release. NPY infused into stalk-ME of prazosin-treated monkeys stimulated LHRH release.

25 This suggests that NPY is working independently of NE/ PGE2. This suggests that NPY is working independently of NE/ PGE2. 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. 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. Thus, it appears that NPY and NE are working independently to modulate LHRH release. Thus, it appears that NPY and NE are working independently to modulate LHRH release.

26 γ-aminobutyric acid (GABA) This is the major inhibitory neurotransmitter in the CNS and it is found in the hypothalamus. This is the major inhibitory neurotransmitter in the CNS and it is found in the hypothalamus. Early studies, involving infusion of GABA into the CSF in the third ventricle, stimulated LHRH release Early studies, involving infusion of GABA into the CSF in the third ventricle, stimulated LHRH release This was also observed in explants of hypothalamic tissue kept in culture. This was also observed in explants of hypothalamic tissue kept in culture.

27 However, other studies were contradictory. However, other studies were contradictory. A more recent observation is that GABA may change in activity with changing age or developmental status. A more recent observation is that GABA may change in activity with changing age or developmental status. In rats, GABA appears to stimulate LHRH release in juveniles, but becomes inhibitory at puberty. In rats, GABA appears to stimulate LHRH release in juveniles, but becomes inhibitory at puberty. 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). 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).

28 In monkeys (using the push-pull cannula)… In monkeys (using the push-pull cannula)… GABA tonically inhibits LHRH release before puberty. GABA tonically inhibits LHRH release before puberty. GABA levels in ME are much higher in pre- pubertal animals and levels drop by mid-puberty GABA levels in ME are much higher in pre- pubertal animals and levels drop by mid-puberty

29 Another neurotransmitter found in the ME region is glutamate. Another neurotransmitter found in the ME region is glutamate. It is an agonist of the excitatory amino acid system, working on NMDA receptors. It is an agonist of the excitatory amino acid system, working on NMDA receptors. It has also been shown to stimulate LHRH release. It has also been shown to stimulate LHRH release. Stimulation of N-methyl-D-aspartate (NMDA) receptors can induce precocious puberty in rats. Stimulation of N-methyl-D-aspartate (NMDA) receptors can induce precocious puberty in rats. There are potential interactions between GABA and glutamate. There are potential interactions between GABA and glutamate.

30 GABA is synthesized from glutamate. GABA is synthesized from glutamate. This raises the possibility that GABA and glutamate levels may be related. This raises the possibility that GABA and glutamate levels may be related. Infusion of antisense oligonucleotides for enzymes involved in the synthesis of GABA from glutamate cause an increase in LHRH in pre-pubertal monkeys. Infusion of antisense oligonucleotides for enzymes involved in the synthesis of GABA from glutamate cause an increase in LHRH in pre-pubertal monkeys. i.e. when GABA synthesis was inhibited, LHRH levels rose. i.e. when GABA synthesis was inhibited, LHRH levels rose.

31 Was this due to a local increase of glutamate, once GABA synthesis was stopped? Was this due to a local increase of glutamate, once GABA synthesis was stopped? Or, did reduction of GABA simply unmask an ongoing glutamate stimulation? Or, did reduction of GABA simply unmask an ongoing glutamate stimulation? Several studies suggest the former. Several studies suggest the former.

32 In pre-pubertal monkeys, the glutamate levels in the stalk ME are very low. In pre-pubertal monkeys, the glutamate levels in the stalk ME are very low. When GABA synthesis is blocked, these levels rise. When GABA synthesis is blocked, these levels rise. However, there are studies that suggest the former may also be occurring. However, there are studies that suggest the former may also be occurring. The probability is that both occur to some extent (and there may be variability between species). The probability is that both occur to some extent (and there may be variability between species).

33 As mentioned before, NO may be playing a role and is an ideal candidate. As mentioned before, NO may be playing a role and is an ideal candidate. Pulsatile LHRH release occurs even when the cell bodies are not present. Pulsatile LHRH release occurs even when the cell bodies are not present. This suggests some form of pre-synaptic stimulation at the neuroterminals of the LHRH neurons. This suggests some form of pre-synaptic stimulation at the neuroterminals of the LHRH neurons. Histological studies have failed to show the presence of pre-synaptic synapses in the right area of the hypothalamus. Histological studies have failed to show the presence of pre-synaptic synapses in the right area of the hypothalamus. 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. 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. This is strong evidence that there is a chemical mediator (i.e.some kind of paracrine signaling), which coordinates the LHRH release.

34 In push-pull cannula experiments, infusion of the NO precursor L-argenine stimulated both NPY and LHRH. In push-pull cannula experiments, infusion of the NO precursor L-argenine stimulated both NPY and LHRH. Infusion of D-argenine had no effect (the dextrorotary form cannot be converted into NO). Infusion of D-argenine had no effect (the dextrorotary form cannot be converted into NO). The enzymes involved in NO synthesis are present in an adjacent area of the hypothalamus. The enzymes involved in NO synthesis are present in an adjacent area of the hypothalamus. This means that NO is available in the area in question. This means that NO is available in the area in question.

35 Finally, glial cells may also be playing a role. Finally, glial cells may also be playing a role. In other systems, glial cells have been shown to modify, or affect the release of neurotransmitters and neurohormones. In other systems, glial cells have been shown to modify, or affect the release of neurotransmitters and neurohormones.

36 In this system, circumstantial evidence suggests this possibility. In this system, circumstantial evidence suggests this possibility. The endogenous pacemaker seems to be located in, or near the neuroterminals of the LHRH cells (and not near the cells bodies). The endogenous pacemaker seems to be located in, or near the neuroterminals of the LHRH cells (and not near the cells bodies). No pre-synaptic synaptic connections have been identified in the area. No pre-synaptic synaptic connections have been identified in the area. Glial cells ARE present around the neuro- terminals. Glial cells ARE present around the neuro- terminals.

37 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. 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. The proposed mechanism for this interaction is that glial cells may release the kallekrein bradykinin. The proposed mechanism for this interaction is that glial cells may release the kallekrein bradykinin. Bradykinin, in turn, would stimulate glutamate release from astrocytes located around the neuroterminals. Bradykinin, in turn, would stimulate glutamate release from astrocytes located around the neuroterminals. This would have an effect on LHRH release from the neuroterminals themselves. This would have an effect on LHRH release from the neuroterminals themselves.

38 LHRH GABA Glu GABA Glu NPY NE NO Glia Portal blood Master Pacemaker?


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