Activation of Gastrin‐releasing Peptide Receptors in the Lumbosacral Spinal Cord is Required for Ejaculation in Male Rats

Introduction.  Ejaculation is a complex reflex mediated by a spinal ejaculation generator located in the lumbosacral spinal cord and consisting of a population of lumbar spinothalamic (LSt) neurons. LSt neurons and their intraspinal axonal projections contain several neuropeptides, including gastrin‐releasing peptide (GRP). Aim.  To test the hypothesis that GRP is critically involved in mediating ejaculation by acting in autonomic and motor areas of the lumbosacral spinal cord, utilizing a physiological paradigm to investigate ejaculatory reflexes in isolation of supraspinal inputs. Methods.  Dual immunohistochemistry for GRP and galanin was performed to investigate co‐expression of GRP in LSt cells of control male rats. Next, anesthetized, spinalized male rats received intrathecal infusions of either GRP antagonist RC‐3095 (0, 10, or 20 nmol/10 µL) or GRP (0, 0.2, 0.5 nmol/10 µL). Ejaculatory reflexes were induced by electrical stimulation of the dorsal penile nerve (DPN) which reliably triggers rhythmic increases in seminal vesicle pressure (SVP) and contractions of the bulbocavernosus muscle (BCM), indicative of the emission and expulsion phases of ejaculation, respectively. Main Outcome Measures.  GRP in LSt cells was expressed as percentages of co‐expression. SVP and electromyographic recording (EMG) of BCM activity following drug treatment and DPN stimulation were recorded and analyzed for numbers of SVP increases, BCM events and bursts. Results.  GRP was exclusively expressed in LSt cells and axons. Intrathecal infusion of RC‐3095, but not saline, blocked SVP increases and BCM bursting induced by DPN stimulation. Intrathecal infusions of GRP, but not saline, triggered SVP increases and BCM bursting in 43–66% of animals and facilitated SVP increases and BCM bursting induced by subthreshold DPN stimulation in all animals. Conclusion.  These data support a critical role for GRP for control of the emission and expulsion phases of ejaculation in male rats by acting in LSt target areas in the lumbosacral spinal cord. Kozyrev N, Lehman MN, and Coolen LM. Activation of gastrin‐releasing peptide receptors in the lumbosacral spinal cord is required for ejaculation in male rats. J Sex Med 2012;9:1303–1318.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91163/1/j.1743-6109.2012.02688.x.pd

Spinal Cord Injury Causes Reduction of Galanin and Gastrin Releasing Peptide mRNA Expression in the Spinal Ejaculation Generator of Male Rats

Spinal cord injury (SCI) in men is commonly associated with sexual dysfunction, including anejaculation, and chronic mid-thoracic contusion injury in male rats also impairs ejaculatory reflexes. Ejaculation is controlled by a spinal ejaculation generator consisting of a population of lumbar spinothalamic (LSt) neurons that control ejaculation through release of four neuropeptides including galanin and gastrin releasing peptide (GRP) onto lumbar and sacral autonomic and motor nuclei. It was recently demonstrated that spinal contusion injury in male rats caused reduction of GRP-immunoreactivity, but not galanin-immunoreactivity in LSt cells, indicative of reduced GRP peptide levels, but inconclusive results for galanin. The current study further tests the hypothesis that contusion injury causes a disruption of GRP and galanin mRNA in LSt cells. Male rats received mid-thoracic contusion injury and galanin and GRP mRNA were visualized 8 weeks later in the lumbar spinal cord using fluorescent in situ hybridization. Spinal cord injury significantly reduced GRP and galanin mRNA in LSt cells. Galanin expression was higher in LSt cells compared to GRP. However, expression of the two transcripts were positively correlated in LSt cells in both sham and SCI animals, suggesting that expression for the two neuropeptides may be co-regulated. Immunofluorescent visualization of galanin and GRP peptides demonstrated a significant reduction in GRP-immunoreactivity, but not galanin in LSt cells, confirming the previous observations. In conclusion, SCI reduced GRP and galanin expression in LSt cells with an apparent greater impact on GRP peptide levels. GRP and galanin are both essential for triggering ejaculation and thus such reduction may contribute to ejaculatory dysfunction following SCI in rats

Chronic Contusion Spinal Cord Injury Impairs Ejaculatory Reflexes in Male Rats: Partial Recovery by Systemic Infusions of Dopamine D3 Receptor Agonist 7OHDPAT

Chronic spinal cord injury (SCI) causes major disruption of ejaculatory function in men. Ejaculation is a reflex and the spinal generator for ejaculatory reflexes in the rat has been located in the lumbosacral spinal cord. The effects of SCI on the rat spinal ejaculation generator and ejaculatory reflexes remain understudied. The first goal of the current study was to establish the effects of chronic SCI on the function of the spinal ejaculation generator. Male rats received a contusion injury of the spinal cord at spinal level T6?T7. Ejaculatory reflexes elicited by electrical stimulation of the dorsal penile nerve (DPN) were evaluated in injured and control rats at 4?6 weeks following SCI. SCI males demonstrated significant reductions in bursting of the bulbocavernosus muscle (BCM), an indicator for expulsion phase of ejaculation, and in seminal vesicle pressure (SVP) increases, an indicator for the emission phase of ejaculation, following DPN stimulation. Thus, contusion SCI resulted in long-term impairment of ejaculatory reflexes. The D3 agonist 7-hydroxy-2-(di-N-propylamino) tetralin (7OHDPAT) facilitates ejaculation in spinal cord intact rats, thus the second goal of the current study was to test whether subcutaneous infusions of 7OHDPAT can facilitate ejaculatory reflexes in rats with chronic SCI. Male rats received a contusion injury at T6?T7 and effects of systemic administration of 7OHDPAT (1?mg/kg) were tested 4?5 weeks following injury. Results showed that 7OHDPAT administration facilitated ejaculatory reflexes in SCI males with or without DPN stimulation, provided that supraspinal inputs to the lumbar cord were severed by transection just prior to evaluating the reflex. Thus, 7OHDPAT administration in SCI males was able to overcome the detrimental effects of SCI on ejaculatory reflexes.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140172/1/neu.2015.4232.pd

Chronic contusion spinal cord injury impairs ejaculatory reflexes in male rats: Partial recovery by systemic infusions of dopamine D3 receptor agonist 7OHDPAT

Chronic spinal cord injury (SCI) causes major disruption of ejaculatory function in men. Ejaculation is a reflex and the spinal generator for ejaculatory reflexes in the rat has been located in the lumbosacral spinal cord. The effects of SCI on the rat spinal ejaculation generator and ejaculatory reflexes remain understudied. The first goal of the current study was to establish the effects of chronic SCI on the function of the spinal ejaculation generator. Male rats received a contusion injury of the spinal cord at spinal level T6-T7. Ejaculatory reflexes elicited by electrical stimulation of the dorsal penile nerve (DPN) were evaluated in injured and control rats at 4-6 weeks following SCI. SCI males demonstrated significant reductions in bursting of the bulbocavernosus muscle (BCM), an indicator for expulsion phase of ejaculation, and in seminal vesicle pressure (SVP) increases, an indicator for the emission phase of ejaculation, following DPN stimulation. Thus, contusion SCI resulted in long-term impairment of ejaculatory reflexes. The D3 agonist 7-hydroxy-2-(di-N-propylamino) tetralin (7OHDPAT) facilitates ejaculation in spinal cord intact rats, thus the second goal of the current study was to test whether subcutaneous infusions of 7OHDPAT can facilitate ejaculatory reflexes in rats with chronic SCI. Male rats received a contusion injury at T6-T7 and effects of systemic administration of 7OHDPAT (1 mg/kg) were tested 4-5 weeks following injury. Results showed that 7OHDPAT administration facilitated ejaculatory reflexes in SCI males with or without DPN stimulation, provided that supraspinal inputs to the lumbar cord were severed by transection just prior to evaluating the reflex. Thus, 7OHDPAT administration in SCI males was able to overcome the detrimental effects of SCI on ejaculatory reflexes

Sex differences and effects of prenatal exposure to excess testosterone on ventral tegmental area dopamine neurons in adult sheep

Prenatal testosterone (T) excess in sheep results in a wide array of reproductive neuroendocrine deficits and alterations in motivated behavior. The ventral tegmental area (VTA) plays a critical role in reward and motivated behaviors and is hypothesised to be targeted by prenatal T. Here we report a sex difference in the number VTA dopamine cells in the adult sheep, with higher numbers of tyrosine hydroxylase (TH)‐immunoreactive (‐ir) cells in males than females. Moreover, prenatal exposure to excess T during either gestational days 30–90 or 60–90 resulted in increased numbers of VTA TH‐ir cells in adult ewes compared to control females. Stereological analysis confirmed significantly greater numbers of neurons in the VTA of males and prenatal T‐treated ewes, which was primarily accounted for by greater numbers of TH‐ir cells. In addition, immunoreactivity for TH in the cells was denser in males and prenatal T‐treated females, suggesting that sex differences and prenatal exposure to excess T affects both numbers of cells expressing TH and the protein levels within dopamine cells. Sex differences were also noted in numbers of TH‐ir cells in the substantia nigra, with more cells in males than females. However, prenatal exposure to excess T did not affect numbers of TH‐ir cells in the substantia nigra, suggesting that this sex difference is organised independently of prenatal actions of T. Together, these results demonstrate sex differences in the sheep VTA dopamine system which are mimicked by prenatal treatment with excess T.We report a sex difference in ventral tegmental area (VTA) dopamine cells in the adult sheep with higher numbers of tyrosine hydroxylase (TH)‐immunoreactive cells in males than females. Moreover, prenatal exposure to excess T during gestational days 30–90 or 60–90 caused increased numbers of VTA TH‐immunoreactive cells in adult ewes compared to control females. Sex differences were also demonstrated in the substantia nigra, but prenatal T had no effect on TH in this area. Results indicate that sex differences and prenatal exposure to excess T affects both numbers of cells expressing TH and the protein levels in the VTA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111123/1/ejn12871.pd

A Pivotal Role of Lumbar Spinothalamic Cells in the Regulation of Ejaculation via Intraspinal Connections

Introduction.  A population of lumbar spinothalamic cells (LSt cells) has been demonstrated to play a pivotal role in ejaculatory behavior and comprise a critical component of the spinal ejaculation generator. LSt cells are hypothesized to regulate ejaculation via their projections to autonomic and motor neurons in the lumbosacral spinal cord. Aim.  The current study tested the hypothesis that ejaculatory reflexes are dependent on LSt cells via projections within the lumbosacral spinal cord. Methods.  Male rats received intraspinal injections of neurotoxin saporin conjugated to substance P analog, previously shown to selectively lesion LSt cells. Two weeks later, males were anesthetized and spinal cords were transected. Subsequently, males were subjected to ejaculatory reflex paradigms, including stimulation of the dorsal penile nerve (DPN), urethrogenital stimulation or administration of D3 agonist 7‐OH‐DPAT. Electromyographic recordings of the bulbocavernosus muscle (BCM) were analyzed for rhythmic bursting characteristic of the expulsion phase of ejaculation. In addition, a fourth commonly used paradigm for ejaculation and erections in unanesthetized, spinal‐intact male rats was utilized: the ex copula reflex paradigm. Main Outcome Measures.  LSt cell lesions were predicted to prevent rhythmic bursting of BCM following DPN, urethral, or pharmacological stimulation, and emissions in the ex copula paradigm. In contrast, LSt cell lesions were not expected to abolish erectile function as measured in the ex copula paradigm. Results.  LSt cell lesions prevented rhythmic contractions of the BCM induced by any of the ejaculatory reflex paradigms in spinalized rats. However, LSt cell lesions did not affect erectile function nor emissions determined in the ex copula reflex paradigm. Conclusions.  These data demonstrate that LSt cells are essential for ejaculatory, but not erectile reflexes, as previously reported for mating animals. Moreover, LSt cells mediate ejaculation via projections within the spinal cord, presumably to autonomic and motor neurons. Staudt MD, Truitt WA, McKenna KE, de Oliveira CVR, Lehman MN, and Coolen LM. A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections. J Sex Med 2012;9:2256–2265.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93690/1/j.1743-6109.2011.02574.x.pd

Evidence that dopamine acts via Kisspeptin to Hold GnRH pulse frequency in check in Anestrous Ewes

Recent work has implicated stimulatory kisspeptin neurons in the arcuate nucleus (ARC) as important for seasonal changes in reproductive function in sheep, but earlier studies support a role for inhibitory A15 dopaminergic (DA) neurons in the suppression of GnRH (and LH) pulse frequency in the nonbreeding (anestrous) season. Because A15 neurons project to the ARC, we performed three experiments to test the hypothesis that A15 neurons act via ARC kisspeptin neurons to inhibit LH in anestrus: 1) we used dual immunocytochemistry to determine whether these ARC neurons contain D2 dopamine receptor (D2-R), the receptor responsible for inhibition of LH in anestrus; 2) wetested the ability of local administration of sulpiride, a D2-R antagonist, into theARCto increase LH secretion in anestrus; and 3) we determined whether an antagonist to the kisspeptin receptor could block the increase in LH secretion induced by sulpiride in anestrus. In experiment 1, 40% of this ARC neuronal subpopulation contained D2-R in breeding season ewes, but this increased to approximately 80% in anestrus. In experiment 2, local microinjection of the two highest doses (10 and 50 nmol) of sulpiride into the ARC significantly increased LH pulse frequency to levels 3 times that seen with vehicle injections. Finally, intracerebroventricular infusion of a kisspeptin receptor antagonist completely blocked the increase in LH pulse frequency induced by systemic administration of sulpiride to anestrous ewes. These results support the hypothesis that DA acts to inhibit GnRH (and LH) secretion in anestrus by suppressing the activity of ARC kisspeptin neurons.We thank Heather Bungard and Jennifer Lydon (West Virginia University Food Animal Research Facility) for the care of animals and Paul Harton for his technical assistance in sectioning tissue. We also thank Dr. Al Parlow and the National Hormone and Peptide Program (Torrance, CA) for the reagents used to measure LH and prolactin.http://endo.endojournals.org/am201

Kisspeptin, neurokinin B, and dynorphin cct in the arcuate nucleus to control activity of the GnRH pulse generator in Ewes

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists toGnRHreceptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion.Wethus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles forNKBand dynorphin within theKNDyneural network and raise the possibility that kisspeptin contributes to the control ofGnRHpulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.National Institutes of Health Grants R01-HD039916 and RO1-HD017864.http://press.endocrine.org/journal/endoam201

Natural Reward Experience Alters AMPA and NMDA Receptor Distribution and Function in the Nucleus Accumbens

Natural reward and drugs of abuse converge upon the mesolimbic system which mediates motivation and reward behaviors. Drugs induce neural adaptations in this system, including transcriptional, morphological, and synaptic changes, which contribute to the development and expression of drug-related memories and addiction. Previously, it has been reported that sexual experience in male rats, a natural reward behavior, induces similar neuroplasticity in the mesolimbic system and affects natural reward and drug-related behavior. The current study determined whether sexual experience causes long-lasting changes in mating, or ionotropic glutamate receptor trafficking or function in the nucleus accumbens (NAc), following 3 different reward abstinence periods: 1 day, 1 week, or 1 month after final mating session. Male Sprague Dawley rats mated during 5 consecutive days (sexual experience) or remained sexually naïve to serve as controls. Sexually experienced males displayed facilitation of initiation and performance of mating at each time point. Next, intracellular and membrane surface expression of N-methyl-D-aspartate (NMDA: NR1 subunit) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA: GluA1, GluA2 subunits) receptors in the NAc was determined using a bis(sulfosuccinimidyl)suberate (BS3) protein cross-linking assay followed by Western Blot analysis. NR1 expression was increased at 1 day abstinence both at surface and intracellular, but decreased at surface at 1 week of abstinence. GluA2 was increased intracellularly at 1 week and increased at the surface after 1 month of abstinence. Finally, whole-cell patch clamp electrophysiological recordings determined reduced AMPA/NMDA ratio of synaptic currents in NAc shell neurons following stimulation of cortical afferents in sexually experienced males after all reward abstinence periods. Together, these data show that sexual experience causes long-term alterations in glutamate receptor expression and function in the NAc. Although not identical, this sex experience-induced neuroplasticity has similarities to that caused by psychostimulants, suggesting common mechanisms for reinforcement of natural and drug reward

Activation of galanin and cholecystokinin receptors in the lumbosacral spinal cord is required for ejaculation in male rats

The spinal ejaculation generator is comprised of lumbar spinothalamic (LSt) cells and their axonal projections to autonomic and motor neurons in the lumbosacral spinal cord. LSt cells regulate ejaculatory reflexes by release of neuropeptides that are co‐expressed in their axons, as previously demonstrated for gastrin‐releasing peptide and enkephalin. Here, the role of two other neuropeptides co‐expressed in LSt cells for ejaculatory reflexes is demonstrated: galanin and cholecystokinin (CCK). Adult male rats were anesthetized, spinalized, and received intrathecal infusions of galanin receptor antagonist Galantide (1 or 10 nmol) or CCK receptor antagonist proglumide (71 or 714 nmol). The dorsal penile nerve (DPN) was electrically stimulated to trigger ejaculatory reflexes and seminal vesicle pressure (SVP) and rhythmic contractions of the bulbocavernosus muscle (BCM) were analyzed as parameters of emission and expulsion respectively. Treatment with galanin or CCK antagonists significantly reduced SVP increases and BCM bursting, demonstrating that galanin and CCK are required for ejaculation. Next, anesthetized, spinalized males received intrathecal infusions of galanin (0.15 or 0.3 nmol) or CCK(26–33) (4.35 nmol) and effects on subthreshold DPN stimulations were determined. Intrathecal infusions of galanin or CCK facilitated ejaculatory reflexes induced by subthreshold DPN stimulation in all animals, but did not trigger ejaculatory reflexes in the absence of DPN stimulation. Together, these results demonstrate that galanin and CCK both act in the spinal ejaculation generator to regulate ejaculation. However, effects of galanin and CCK were dependent on DPN stimulation, suggesting that these neuropeptides may act in concert with other LSt co‐expressed neuropeptides.The spinal ejaculation generator is comprised of lumbar spinothalamic (LSt) cells and their axonal projections to autonomic andmotor neurons in the lumbosacral spinal cord. LSt cells express and release several neuropeptides, including galanin and cholecystokinin (CCK). This study demonstrates that galanin and CCK both act in the spinal ejaculation generator to regulate the ejaculatory reflex.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136351/1/ejn13515_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136351/2/ejn13515.pd