Rostral ventromedial medulla
Encyclopedia
The rostral ventromedial medulla (RVM), is a group of neurons located close to the midline on the floor of the medulla (myelencephalon
). The rostral ventromedial medulla sends descending inhibitory and excitatory fibers to the dorsal horn spinal cord
neurons.
There are 3 categories of neurons in the RVM. On-cells, off-cells, and neutral cells. They are characterized by their response to nociceptive input. Off-cells show a transitory decrease in firing rate right before a nociceptive reflex, and are theorized to be inhibitory. Activation of off-cells, either by morphine or any other means, results in antinociception. On-cells show a burst of activity immediately preceding nociceptive input, and are theorized to be contributing to the excitatory drive. Neutral cells show no response to nociceptive input.
expressing neurons in the RVM with a dermorphin-saporin
conjugate eliminated the allodynia
and hyperalgesia
caused by a nerve injury. Treatment with the dermorphin-saporin conjugate did not alter baseline pain thresholds, or affect sensitivity in the first 5–10 days after nerve injury. This suggests that the RVM contributes to the persistent pathology caused by nerve injury.
Further research determined that a large majority of μ-opioid expressing neurons also expressed CCK2
receptors. Microinjection in the RVM with either a CCK-saporin or dermorphin-saporin conjugate eliminated neurons expressing either receptor. Injection of the CCK-saporin conjugate also reversed allodynia and hyperalgesia in a nerve injury model, producing the same results as the dermorphin-saporin conjugate. This destruction of neurons was relatively specific, as less than 10% of neurons in the RVM were destroyed. This suggests that the targeted neurons are the ones responsible for maintaining chronic neuropathic pain states, and that the observed effect wasn't due to diffuse destruction of RVM neurons.
In addition, lidocaine
microinjections into the RVM temporarily reversed allodynia and hyperalgesia caused by nerve injury.
To help determine whether the persistent pain state was centrally or peripherally mediated, non-noxious stimuli were applied to the nerve injured limb. In animals receiving vehicle injections into the RVM, there was an increase in FOS
expression in the superficial and deep dorsal horn of the spinal cord, indicating activation of nociceptive neurons. Animals receiving the dermorphin-saporin conjugate into the RVM had significantly less FOS expression. This indicates that a persistent neuropathic pain state is centrally mediated.
, ondansetron
, and a 5-HT7 antagonist, SB-269,970
, were chosen to study.
System or intra-RVM injections of morphine
produced dose-dependent antinociception. Spinal administration
of SB-269970 reduced morphine-induced antinociception, whereas spinal administration of ondansetron had no effects. SB-269970 and ondansetron were then tested for their efficacy in reducing nociceptive responses. Allodynia
and hyperalgesia
were experimentally induced by administration of CCK into the RVM. Spinal administration of SB-269970 had no effect on nociception
, whereas ondansetron completely reversed the effects of CCK injection. Spinal ondansetron also reversed allodynia and hyperalgesia caused by a peripheral nerve injury
. Taken together, these findings indicate a role for 5-HT7 receptors in opioid induced antinociception, and a role for 5-HT3 in pro-nociceptive facilitation.
One limiting factor is that SB-269970 was also found to be a potent α2-adrenergic
antagonist. Since the study using SB-269970 did not use a
α2-adrenergic antagonist as a control, it is possible that some of the effects of SB-269970 are from its adrenergic effects.
(SP). Microinjections of SP into the RVM resulted in transient antinociception to noxious heat stimuli, but not mechanical stimuli. Pretreatment with a neurokinin 1 (NK1) antagonist
prevented the antinociception induced by SP injection, but the NK1 antagonist had no effects on pain threshold by itself. To test the effects of an NK1 antagonist during injury states, an NK1 antagonist was microinjected into the RVM after application of Freund's Complete Adjuvant
(CFA), a chemical used for inflammation models. Administration of the NK1 antagonist reversed the heat hyperalgesia caused by CFA. In contrast, the administration of an NK1 antagonist further increased the tactile hyperalgesia induced by CFA. However, the NK1 antagonist did prevent some tactile hyperalgesia induced by a different compound, capsaicin
. In yet another induced injury model using mustard oil
(a TRPA1
agonist), NK1 antagonists did not affect thermal or tactile hyperalgesia.
In contrast to the study above, another group of researchers found that microinjection of SP into the RVM resulted in transient thermal hyperalgesia, which persisted long-term when continuous infusion pumps were implanted. To look more at the SP-NK1 signaling, they performed Western Blots of RVM slices, looking for NK1 receptor expression. NK1 receptor expression was increased from 2 hours to 3 days after administration of CFA.
NK1 agonism induced hypersensitivity is dependent on 5-HT3 receptors, and modulated by GABAA and NMDA receptors as well. Animals were pretreated with spinally administered Y-25130 or ondansetron
, both 5-HT3 antagonists, before having RVM injections of SP. Both Y-25130 and ondansetron inhibited SP induced thermal hyperalgesia. GABAA receptor involvement was demonstrated by intrathecal
administration of gabazine
, a GABAA antagonist, in animals receiving continuous infusions of SP into the RVM. Gabazine treatment completely reversed the thermal hyperalgesia. The mechanism behind GABA involvement was investigated using ""in vitro"" recordings from animals treated with continuous infusions of SP or saline into the RVM. In SP treated neurons, GABA evoked depolarization, while in saline treated neurons it caused hyperpolarization. "These results suggest that descending facilitation induced by RVM SP administration produces GABAA receptor-evoked depolarization and an increase in excitation of dorsal horn neurons." Next, a GABA A agonist, muscimol
was tested in conjuncture with SP. Intrathecal muscimol significantly enhanced SP-induced hypersensitivity, which was blocked by intrathecal gabazine. Next the researchers looked at threonine phosphorylation of NKCC1 proteins, which are an isoform of the Na-K-Cl cotransporter. Phosphorylation of these proteins results in increase activity of the cotransporter. Chronic administration of RVM SP or acute SP combined with intrathecal muscimol resulted in significantly higher levels of phosphorylated NKCC1.
s in non-inflammatory noxious stimuli was examined. The injury model consisted on two injections of acidic saline (pH = 4.0), and was designed to model non-inflammatory muscular pain. Intra-RVM administration of AP5 or MK-801, NMDA receptor antagonists, resulted in a reversal of the mechanical sensitivity induced by the acidic saline.
Behavioral hyperalgesia
in inflammatory pain states is closely correlated with phosphorylation of spinal NMDA receptors. To find out more about the role of NMDA receptors in RVM pain facilitation, intrathecal
MK-801 was administered before a RVM SP
injection. Pretreatment with MK-801 significantly reduced SP induced hyperalgesia. Intrathecal MK-801 also blocked hyperalgesia resulting from continuous SP infusions. SP also increased the phosphorylation of the NR1 subunit of NMDA receptors.
In order to find out the relationship between GABA
, NMDA, and SP, MK-801 was administered intrathecally to determine the effect on muscimol potentiation of SP hyperalgesia. MK-801 reduced the exaggeration of SP hyperalgesia induced by muscimol. Also, low doses of SP and intrathecal muscimol increased the expression of phosphorylated NR1 subunits of NMDA receptors. Intrathecal gabazine treatment before muscimol blocked the increase in phosphorylated NR1 expression.
, a P1 and P2 agonist, whereas neutral cells were inhibited. However, on-cells and off-cells differed in their response to P2X
and P2Y agonists
.
On cells displayed a greater response to P2X agonists vs P2Y agonists. For example, α,β-methylene ATP, a P2X agonist, activated all on cells, whereas 2-methylthio-ATP, a P2Y agonist, only activated 60% of on cells tested. All on cells showed a response to a non-specific P2 agonist, uridine triphosphate (UTP). Activation of on cells by ATP was reversed by using the P2 antagonists suramin
and pyridoxal-phosphate-6-azophenyl-2′,4′disulphonic acid (PPADS
), but not with the P2Y antagonist MRS2179.
In contrast, off-cells were more responsive to P2Y agonists. 2-methylthio-ATP activated all off-cells, whereas α,β-methylene ATP, a P2X agonist, only activated one-third of off-cells. Off-cells were also activated by UTP, but lacked any response to adenosine, a P1 agonist. Activation of off-cells by ATP was inhibited by suramin, PPADS, and MRS2179.
Neutral cells are inhibited by adenosine, a P1 agonist, whereas on-cells and off-cells lack a response to adenosine.
Histological staining by another research group examined the distribution of purinergic receptor subtypes throughout the RVM. P1, P2X1, and P2X3 all showed moderate labeling density, with slightly greater densities observed in the nucleus raphe magnus
and the raphe pallidus. In contrast, P2Y1 showed lower levels of labeling. P1 and P2Y1 were shown to be co-localized, as well as P2X1 and P2Y1. Presence of the raphe nuclei
in the RVM also led to staining for tryptophan hydroxylase
(TPH), a marker for serotonin
(5-HT) positive neurons, and looking for co-localization of 5-HT neurons with purinergic receptors. Only about 10% of RVM neurons were TPH positive, but of those labeled for TPH, a large majority were co-labeled with purinergic antibodies. 55% of TPH+ neurons stained for P1, 63% for P2X1, 64% for P2X3, and 70% P2Y1.
Myelencephalon
The myelencephalon is categorized as a secondary vesicle in the development of the central nervous system. The prefix "myelen" is derived from Greek for medulla...
). The rostral ventromedial medulla sends descending inhibitory and excitatory fibers to the dorsal horn spinal cord
Spinal cord
The spinal cord is a long, thin, tubular bundle of nervous tissue and support cells that extends from the brain . The brain and spinal cord together make up the central nervous system...
neurons.
There are 3 categories of neurons in the RVM. On-cells, off-cells, and neutral cells. They are characterized by their response to nociceptive input. Off-cells show a transitory decrease in firing rate right before a nociceptive reflex, and are theorized to be inhibitory. Activation of off-cells, either by morphine or any other means, results in antinociception. On-cells show a burst of activity immediately preceding nociceptive input, and are theorized to be contributing to the excitatory drive. Neutral cells show no response to nociceptive input.
Involvement in Neuropathic pain
Research has shown the RVM to be important in the maintenance of neuropathic pain. Ablation of μ-opioidMu Opioid receptor
The μ-opioid receptors are a class of opioid receptors with high affinity for enkephalins and beta-endorphin but low affinity for dynorphins. They are also referred to as μ opioid peptide receptors. The prototypical μ receptor agonist is the opium alkaloid morphine; μ refers to morphine...
expressing neurons in the RVM with a dermorphin-saporin
Saporin
Saporin is a protein that is useful in biological research applications, especially studies of behavior. Saporin is a so-called ribosome inactivating protein , due to its N-glycosidase activity, from the seeds of Saponaria officinalis . It was first described by Fiorenzo Stirpe and his colleagues...
conjugate eliminated the allodynia
Allodynia
Allodynia is a pain due to a stimulus which does not normally provoke pain. Temperature or physical stimuli can provoke allodynia, and it often occurs after injury to a site...
and hyperalgesia
Hyperalgesia
Hyperalgesia is an increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves. Temporary increased sensitivity to pain also occurs as part of sickness behavior, the evolved response to infection.-Types:...
caused by a nerve injury. Treatment with the dermorphin-saporin conjugate did not alter baseline pain thresholds, or affect sensitivity in the first 5–10 days after nerve injury. This suggests that the RVM contributes to the persistent pathology caused by nerve injury.
Further research determined that a large majority of μ-opioid expressing neurons also expressed CCK2
Cholecystokinin
Cholecystokinin is a peptide hormone of the gastrointestinal system responsible for stimulating the digestion of fat and protein...
receptors. Microinjection in the RVM with either a CCK-saporin or dermorphin-saporin conjugate eliminated neurons expressing either receptor. Injection of the CCK-saporin conjugate also reversed allodynia and hyperalgesia in a nerve injury model, producing the same results as the dermorphin-saporin conjugate. This destruction of neurons was relatively specific, as less than 10% of neurons in the RVM were destroyed. This suggests that the targeted neurons are the ones responsible for maintaining chronic neuropathic pain states, and that the observed effect wasn't due to diffuse destruction of RVM neurons.
In addition, lidocaine
Lidocaine
Lidocaine , Xylocaine, or lignocaine is a common local anesthetic and antiarrhythmic drug. Lidocaine is used topically to relieve itching, burning and pain from skin inflammations, injected as a dental anesthetic or as a local anesthetic for minor surgery.- History :Lidocaine, the first amino...
microinjections into the RVM temporarily reversed allodynia and hyperalgesia caused by nerve injury.
To help determine whether the persistent pain state was centrally or peripherally mediated, non-noxious stimuli were applied to the nerve injured limb. In animals receiving vehicle injections into the RVM, there was an increase in FOS
C-Fos
In the field of molecular biology and Genetics, c-Fos is a protein encoded by the FOS gene.-Structure and function:c-Fos is a cellular proto-oncogene belonging to the immediate early gene family of transcription factors. c-Fos has a leucine-zipper DNA binding domain, and a transactivation domain at...
expression in the superficial and deep dorsal horn of the spinal cord, indicating activation of nociceptive neurons. Animals receiving the dermorphin-saporin conjugate into the RVM had significantly less FOS expression. This indicates that a persistent neuropathic pain state is centrally mediated.
Role of Serotonin in Pain Modulation
Serotonin receptors have been hypothesized to play a bidirectional role in the modulation of pain. Based on previous experiments, a 5-HT3 antagonistAntagonist
An antagonist is a character, group of characters, or institution, that represents the opposition against which the protagonist must contend...
, ondansetron
Ondansetron
Ondansetron is a serotonin 5-HT3 receptor antagonist used mainly as an antiemetic , often following chemotherapy. Its effects are thought to be on both peripheral and central nerves...
, and a 5-HT7 antagonist, SB-269,970
SB-269,970
SB-269,970 is a drug and research chemical developed by GlaxoSmithKline used in scientific studies. It was previously believed to act as a selective 5-HT7 receptor antagonist , but a subsequent discovery showed that it also potently blocks the α2-adrenergic receptor...
, were chosen to study.
System or intra-RVM injections of morphine
Morphine
Morphine is a potent opiate analgesic medication and is considered to be the prototypical opioid. It was first isolated in 1804 by Friedrich Sertürner, first distributed by same in 1817, and first commercially sold by Merck in 1827, which at the time was a single small chemists' shop. It was more...
produced dose-dependent antinociception. Spinal administration
Intrathecal
Intrathecal is an adjective that refers to something introduced into or occurring in the space under the arachnoid membrane of the brain or spinal cord...
of SB-269970 reduced morphine-induced antinociception, whereas spinal administration of ondansetron had no effects. SB-269970 and ondansetron were then tested for their efficacy in reducing nociceptive responses. Allodynia
Allodynia
Allodynia is a pain due to a stimulus which does not normally provoke pain. Temperature or physical stimuli can provoke allodynia, and it often occurs after injury to a site...
and hyperalgesia
Hyperalgesia
Hyperalgesia is an increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves. Temporary increased sensitivity to pain also occurs as part of sickness behavior, the evolved response to infection.-Types:...
were experimentally induced by administration of CCK into the RVM. Spinal administration of SB-269970 had no effect on nociception
Nociception
Nociception is defined as "the neural processes of encoding and processing noxious stimuli." It is the afferent activity produced in the peripheral and central nervous system by stimuli that have the potential to damage tissue...
, whereas ondansetron completely reversed the effects of CCK injection. Spinal ondansetron also reversed allodynia and hyperalgesia caused by a peripheral nerve injury
Nerve injury
Nerve injury is injury to nervous tissue. There is no single classification system that can describe all the many variations of nerve injury. Most systems attempt to correlate the degree of injury with symptoms, pathology and prognosis...
. Taken together, these findings indicate a role for 5-HT7 receptors in opioid induced antinociception, and a role for 5-HT3 in pro-nociceptive facilitation.
One limiting factor is that SB-269970 was also found to be a potent α2-adrenergic
Alpha-2 adrenergic receptor
The alpha-2 adrenergic receptor is a G protein-coupled receptor associated with the Gi heterotrimeric G-protein. It consists of three highly homologous subtypes, including α2A-, α2B-, and α2C-adrenergic. Some species other than humans express a fourth α2D-adrenergic receptor as well...
antagonist. Since the study using SB-269970 did not use a
α2-adrenergic antagonist as a control, it is possible that some of the effects of SB-269970 are from its adrenergic effects.
Effects of Substance P and Neurokinin 1 receptors
The RVM contains high levels of both the neurokinin 1 receptor and its endogenous ligand, Substance PSubstance P
In the field of neuroscience, substance P is a neuropeptide: an undecapeptide that functions as a neurotransmitter and as a neuromodulator. It belongs to the tachykinin neuropeptide family. Substance P and its closely related neuropeptide neurokinin A are produced from a polyprotein precursor...
(SP). Microinjections of SP into the RVM resulted in transient antinociception to noxious heat stimuli, but not mechanical stimuli. Pretreatment with a neurokinin 1 (NK1) antagonist
Antagonist
An antagonist is a character, group of characters, or institution, that represents the opposition against which the protagonist must contend...
prevented the antinociception induced by SP injection, but the NK1 antagonist had no effects on pain threshold by itself. To test the effects of an NK1 antagonist during injury states, an NK1 antagonist was microinjected into the RVM after application of Freund's Complete Adjuvant
Freund's adjuvant
Freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immunopotentiator . The complete form, Freund's Complete Adjuvant, is composed of inactivated and dried mycobacteria , whereas the incomplete form lacks the mycobacterial components...
(CFA), a chemical used for inflammation models. Administration of the NK1 antagonist reversed the heat hyperalgesia caused by CFA. In contrast, the administration of an NK1 antagonist further increased the tactile hyperalgesia induced by CFA. However, the NK1 antagonist did prevent some tactile hyperalgesia induced by a different compound, capsaicin
Capsaicin
Capsaicin 2CHCH=CH4CONHCH2C6H3-4--3- ) is the active component of chili peppers, which are plants belonging to the genus Capsicum. It is an irritant for mammals, including humans, and produces a sensation of burning in any tissue with which it comes into contact...
. In yet another induced injury model using mustard oil
Mustard oil
The term mustard oil is used for three different oils that are made from mustard seeds:*A fatty vegetable oil resulting from pressing the seeds,...
(a TRPA1
TRPA1
Transient receptor potential cation channel, subfamily A, member 1, also known as TRPA1, is a protein which in humans is encoded by the TRPA1 gene....
agonist), NK1 antagonists did not affect thermal or tactile hyperalgesia.
In contrast to the study above, another group of researchers found that microinjection of SP into the RVM resulted in transient thermal hyperalgesia, which persisted long-term when continuous infusion pumps were implanted. To look more at the SP-NK1 signaling, they performed Western Blots of RVM slices, looking for NK1 receptor expression. NK1 receptor expression was increased from 2 hours to 3 days after administration of CFA.
NK1 agonism induced hypersensitivity is dependent on 5-HT3 receptors, and modulated by GABAA and NMDA receptors as well. Animals were pretreated with spinally administered Y-25130 or ondansetron
Ondansetron
Ondansetron is a serotonin 5-HT3 receptor antagonist used mainly as an antiemetic , often following chemotherapy. Its effects are thought to be on both peripheral and central nerves...
, both 5-HT3 antagonists, before having RVM injections of SP. Both Y-25130 and ondansetron inhibited SP induced thermal hyperalgesia. GABAA receptor involvement was demonstrated by intrathecal
Intrathecal
Intrathecal is an adjective that refers to something introduced into or occurring in the space under the arachnoid membrane of the brain or spinal cord...
administration of gabazine
Gabazine
Gabazine is a drug that acts as an antagonist at GABAA receptors. It is used in scientific research and has no role in medicine, as it would be expected to produce convulsions if used in humans....
, a GABAA antagonist, in animals receiving continuous infusions of SP into the RVM. Gabazine treatment completely reversed the thermal hyperalgesia. The mechanism behind GABA involvement was investigated using ""in vitro"" recordings from animals treated with continuous infusions of SP or saline into the RVM. In SP treated neurons, GABA evoked depolarization, while in saline treated neurons it caused hyperpolarization. "These results suggest that descending facilitation induced by RVM SP administration produces GABAA receptor-evoked depolarization and an increase in excitation of dorsal horn neurons." Next, a GABA A agonist, muscimol
Muscimol
Muscimol is the major psychoactive alkaloid present in many mushrooms of the Amanita genus. Unlike psilocin , which is a serotonergic psychedelic and agonist for the 5-HT2A receptor set, muscimol is a potent, selective agonist for the GABAA receptor set and is a deliriant as a opposed...
was tested in conjuncture with SP. Intrathecal muscimol significantly enhanced SP-induced hypersensitivity, which was blocked by intrathecal gabazine. Next the researchers looked at threonine phosphorylation of NKCC1 proteins, which are an isoform of the Na-K-Cl cotransporter. Phosphorylation of these proteins results in increase activity of the cotransporter. Chronic administration of RVM SP or acute SP combined with intrathecal muscimol resulted in significantly higher levels of phosphorylated NKCC1.
Involvement of NMDA receptors
The role of NMDA receptorNMDA receptor
The NMDA receptor , a glutamate receptor, is the predominant molecular device for controlling synaptic plasticity and memory function....
s in non-inflammatory noxious stimuli was examined. The injury model consisted on two injections of acidic saline (pH = 4.0), and was designed to model non-inflammatory muscular pain. Intra-RVM administration of AP5 or MK-801, NMDA receptor antagonists, resulted in a reversal of the mechanical sensitivity induced by the acidic saline.
Behavioral hyperalgesia
Hyperalgesia
Hyperalgesia is an increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves. Temporary increased sensitivity to pain also occurs as part of sickness behavior, the evolved response to infection.-Types:...
in inflammatory pain states is closely correlated with phosphorylation of spinal NMDA receptors. To find out more about the role of NMDA receptors in RVM pain facilitation, intrathecal
Intrathecal
Intrathecal is an adjective that refers to something introduced into or occurring in the space under the arachnoid membrane of the brain or spinal cord...
MK-801 was administered before a RVM SP
Substance P
In the field of neuroscience, substance P is a neuropeptide: an undecapeptide that functions as a neurotransmitter and as a neuromodulator. It belongs to the tachykinin neuropeptide family. Substance P and its closely related neuropeptide neurokinin A are produced from a polyprotein precursor...
injection. Pretreatment with MK-801 significantly reduced SP induced hyperalgesia. Intrathecal MK-801 also blocked hyperalgesia resulting from continuous SP infusions. SP also increased the phosphorylation of the NR1 subunit of NMDA receptors.
In order to find out the relationship between GABA
Gabâ
Gabâ or gabaa, for the people in many parts of the Philippines), is the concept of a non-human and non-divine, imminent retribution. A sort of negative karma, it is generally seen as an evil effect on a person because of their wrongdoings or transgressions...
, NMDA, and SP, MK-801 was administered intrathecally to determine the effect on muscimol potentiation of SP hyperalgesia. MK-801 reduced the exaggeration of SP hyperalgesia induced by muscimol. Also, low doses of SP and intrathecal muscimol increased the expression of phosphorylated NR1 subunits of NMDA receptors. Intrathecal gabazine treatment before muscimol blocked the increase in phosphorylated NR1 expression.
Purinergic Involvement
On and off cells were both activated by local administration of ATPAdenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
, a P1 and P2 agonist, whereas neutral cells were inhibited. However, on-cells and off-cells differed in their response to P2X
P2X Receptor
P2X receptors are a family of cation-permeable ligand gated ion channels that open in response to the binding of extracellular adenosine 5'-triphosphate . They belong to a larger family of receptors known as the purinergic receptors...
and P2Y agonists
P2Y receptor
P2Y receptors are a family of purinergic receptors, G protein-coupled receptors stimulated by nucleotides such as ATP, ADP, UTP, UDP and UDP-glucose...
.
On cells displayed a greater response to P2X agonists vs P2Y agonists. For example, α,β-methylene ATP, a P2X agonist, activated all on cells, whereas 2-methylthio-ATP, a P2Y agonist, only activated 60% of on cells tested. All on cells showed a response to a non-specific P2 agonist, uridine triphosphate (UTP). Activation of on cells by ATP was reversed by using the P2 antagonists suramin
Suramin
Suramin is a drug developed by Oskar Dressel and Richard Kothe of Bayer, Germany in 1916, and is still sold by Bayer under the brand name Germanin.According to the National Cancer Institute there are no active clinical trials...
and pyridoxal-phosphate-6-azophenyl-2′,4′disulphonic acid (PPADS
PPADS
PPADS is a selective purinergic P2X antagonist. It is able to block contractions of rabbit vas deferens induced by ATP or α,β,methylene-ATP. It appears to be relatively selection for P2X receptors, having no appreciable activity at α1 adrenergic, muscarinic M2 and M3, histamine H1, and adenosine...
), but not with the P2Y antagonist MRS2179.
In contrast, off-cells were more responsive to P2Y agonists. 2-methylthio-ATP activated all off-cells, whereas α,β-methylene ATP, a P2X agonist, only activated one-third of off-cells. Off-cells were also activated by UTP, but lacked any response to adenosine, a P1 agonist. Activation of off-cells by ATP was inhibited by suramin, PPADS, and MRS2179.
Neutral cells are inhibited by adenosine, a P1 agonist, whereas on-cells and off-cells lack a response to adenosine.
Histological staining by another research group examined the distribution of purinergic receptor subtypes throughout the RVM. P1, P2X1, and P2X3 all showed moderate labeling density, with slightly greater densities observed in the nucleus raphe magnus
Nucleus raphe magnus
The nucleus raphes magnus, located directly rostral to the nucleus raphes obscurus, is afferently stimulated from axons in the spinal cord and cerebellum....
and the raphe pallidus. In contrast, P2Y1 showed lower levels of labeling. P1 and P2Y1 were shown to be co-localized, as well as P2X1 and P2Y1. Presence of the raphe nuclei
Raphe nuclei
The raphe nuclei are a moderate-size cluster of nuclei found in the brain stem. Their main function is to release serotonin to the rest of the brain...
in the RVM also led to staining for tryptophan hydroxylase
Tryptophan hydroxylase
Tryptophan hydroxylase is an enzyme involved in the synthesis of the neurotransmitter serotonin. TPH catalyzes the following chemical reactionIt employs one cofactor, iron.- Function :...
(TPH), a marker for serotonin
Serotonin
Serotonin or 5-hydroxytryptamine is a monoamine neurotransmitter. Biochemically derived from tryptophan, serotonin is primarily found in the gastrointestinal tract, platelets, and in the central nervous system of animals including humans...
(5-HT) positive neurons, and looking for co-localization of 5-HT neurons with purinergic receptors. Only about 10% of RVM neurons were TPH positive, but of those labeled for TPH, a large majority were co-labeled with purinergic antibodies. 55% of TPH+ neurons stained for P1, 63% for P2X1, 64% for P2X3, and 70% P2Y1.