Gender-affirming hormone treatment: friend or foe? Long-term follow-up of 755 transgender people.

PURPOSE: Gender-affirming hormone treatment (GAHT) is one of the main demands of transgender and gender diverse (TGD) people, who are usually categorised as transgender assigned-male-at birth (AMAB) and assigned-female-at birth (AFAB). The aim of the study is to investigate the long-term therapeutic management of GAHT, considering hormonal targets, treatment adjustments and GAHT safety. METHODS: A retrospective, longitudinal, observational, multicentre clinical study was carried out. Transgender people, both AMAB and AFAB, were recruited from two Endocrinology Units in Italy (Turin and Modena) between 2005 and 2022. Each subject was managed with specific and personalized follow-up depending on the clinical practice of the Centre. All clinical data routinely collected were extracted, including anthropometric and biochemical parameters, lifestyle habits, GAHT regime, and cardiovascular events. RESULTS: Three-hundred and two transgender AFAB and 453 transgender AMAB were included. Similar follow-up duration (p = 0.974) and visits' number (p = 0.384) were detected between groups. The transgender AFAB group reached therapeutic goals in less time (p = 0.002), fewer visits (p = 0.006) and fewer adjustments of GAHT scheme (p = 0.024). Accordingly, transgender AFAB showed a higher adherence to medical prescriptions compared to transgender AMAB people (p < 0.001). No significantly increased rate of cardiovascular events was detected in both groups. CONCLUSION: Our real-world clinical study shows that transgender AFAB achieve hormone target earlier and more frequently in comparison to transgender AMAB individuals. Therefore, transgender AMAB people may require more frequent check-ups in order to tailor feminizing GAHT and increase therapeutic adherence.

The steroid response to human chorionic gonadotropin (hCG) stimulation in men with Klinefelter syndrome does not change using immunoassay or mass spectrometry.

PURPOSE: Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) was developed in parallel to Immunoassays (IAs) and today is proposed as the "gold standard" for steroid assays. Leydig cells of men with Klinefelter syndrome (KS) are able to respond to human chorionic gonadotropin (hCG) stimulation, even if testosterone (T) production was impaired. The aim was to evaluate how results obtained by IAs and LC-MS/MS can differently impact on the outcome of a clinical research on gonadal steroidogenesis after hCG stimulation. METHODS: A longitudinal, prospective, case-control clinical trial. (clinicaltrial.gov NCT02788136) was carried out, enrolling KS men and healthy age-matched controls, stimulated by hCG administration. Serum steroids were evaluated at baseline and for 5 days after intramuscular injection of 5000 IU hCG using both IAs and LC-MS/MS. RESULTS: 13 KS patients (36 ± 9 years) not receiving T replacement therapy and 14 controls (32 ± 8 years) were enrolled. T, progesterone, cortisol, 17-hydroxy-progesterone (17OHP) and androstenedione, were significantly higher using IAs than LC-MS/MS. IAs and LC-MS/MS showed direct correlation for all five steroids, although the constant overestimation detected by IAs. Either methodology found the same 17OHP and T increasing profile after hCG stimulation, with equal areas under the curves (AUCs). CONCLUSIONS: Although a linearity between IA and LC-MS/MS is demonstrated, LC-MS/MS is more sensitive and accurate, whereas IA shows a constant overestimation of sex steroid levels. This result suggests the need of reference intervals built on the specific assay. This fundamental difference between these two methodologies opens a deep reconsideration of what is needed to improve the accuracy of steroid hormone assays.

Risk behaviours and alcohol in adolescence are negatively associated with testicular volume: results from the Amico-Andrologo survey.

BACKGROUND: Risk factors established during adolescence affect health outcomes in adulthood, although little is known about how adolescent health risk behaviours (HRBs) affect testicular development and reproductive health. OBJECTIVES: To assess prevalence of HRBs among last year high school students; to describe the most prevalent andrological disorders in this cohort; to explore HRBs associated with andrological disorders and investigate factors possibly associated with impaired testicular development in puberty. MATERIALS AND METHODS: The Amico-Andrologo Survey is a permanent nationwide surveillance programme conducted by the Italian Society of Andrology and Sexual Medicine and supported by the Ministry of Health. A nationally representative survey of final-year male high school students was conducted using a validated structured interview (n = 10124) and medical examination (n = 3816). RESULTS: Smoking (32.6%), drinking (80.6%) and use of illegal drugs (46.5%) are common in adolescence. 16.6% of subjects were overweight, 3.1% were underweight and 2.3% were obese. Among sexually active students (60.3%), unprotected sex was very common (48.3%). Only 11.6% had been treated for andrological disorders, despite an abnormal clinical examination in 34.6%. Bilateral testicular hypotrophy (14.0%), varicocoele (27.1%) and phimosis (7.1%) were the most prevalent disorders; 5.1% complained of premature ejaculation and 4.7% had an STI. Underweight and heavy alcohol or drug use were associated with testicular hypotrophy. HRBs emerged as significant predictors of testicular hypotrophy, explaining up to 9.6% of its variance. Limitations include risk of selection bias for voluntary physical examination and recall bias for the self-compiled questionnaire. DISCUSSION: There is an emerging global adverse trend of HRBs in male high school students. A significant proportion of adolescent males with unsuspected andrological disorders engage in behaviours that could impair testicular development. CONCLUSION: Greater attention to the prevention of andrological health in adolescence is needed.

Osteoporosis and male age-related hypogonadism: role of sex steroids on bone (patho)physiology.

Male age-related bone loss is caused, at least in part, by hypogonadism that occurs with advancing age. The study of the effects of sex steroids on bone physiology in men has recently highlighted the central role of estrogens on bone pathophysiology. This review focuses on particular aspects of bone physiology and pathophysiology in aging men, noting both the similarities to and the differences from female counterparts. In particular, the role of sex steroids on bone sexual dimorphism in health and disease has been analyzed.

Human chorionic gonadotropin stimulation gives evidence of differences in testicular steroidogenesis in Klinefelter syndrome, as assessed by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Men with Klinefelter syndrome (KS) show hypergonadotropic hypogonadism, but the pathogenesis of hypotestosteronemia remains unclear. Testicular steroidogenesis in KS men was evaluated over three decades ago after human chorionic gonadotropin (hCG) stimulation, but inconclusive results were obtained. Intriguingly, some recent studies show increased intratesticular testosterone concentrations in men with KS. OBJECTIVE: To analyze serum steroid profile, as a proxy of testicular steroidogenesis, after hCG stimulation in KS compared with control men. DESIGN: A prospective, longitudinal, case-control, clinical trial. METHODS: Thirteen KS patients (36±9 years) not receiving testosterone (TS) replacement therapy and 12 eugonadic controls (32±8 years) were enrolled. Serum steroids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at baseline and for five consecutive days after intramuscular injection of 5000IU hCG. RESULTS: Progesterone (P), 17-hydroxyprogesterone (17OHP), TS, and estradiol (E2) showed a significant increase (P<0.001) after hCG stimulation in both groups. On the contrary, androstenedione (AS) and dehydroepiandrosterone did not increase after hCG stimulation. The 17OHP/P ratio increased in both groups (P<0.001), the TS/AS ratio (17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3) activity) did not increase after hCG in any group, and the E2/TS ratio (aromatase activity) increased significantly in both groups (P=0.009 in KS and P<0.001 in controls). Luteinizing hormone decreased after hCG in both groups (P=0.014 in KS and P<0.001 in controls), whereas follicle-stimulating hormone decreased only in control men (P<0.001). CONCLUSION: This study demonstrates for the first time using LC-MS/MS that Leydig cells of KS men are able to respond to hCG stimulation and that the first steps of steroidogenesis are fully functional. However, the TS production in KS men is impaired, possibly related to reduced hydroxysteroid deydrogenase activity due to an unfavorable intratesticular metabolic state.

FSH treatment of male idiopathic infertility improves pregnancy rate: a meta-analysis.

INTRODUCTION: The aim of this study is to comprehensively evaluate whether FSH administration to the male partner of infertile couples improves pregnancy rate, spontaneously and/or after assisted reproductive techniques (ART). METHODS: Meta-analysis of controlled clinical trials in which FSH was administered for male idiopathic infertility, compared with placebo or no treatment. Randomization was not considered as an inclusion criterion. RESULTS: We found 15 controlled clinical studies (614 men treated with FSH and 661 treated with placebo or untreated). Concerning the type of FSH, eight studies used recombinant FSH, whereas seven studies used purified FSH. Nine studies evaluated spontaneous pregnancy rate, resulting in an overall odds ratio (OR) of about 4.5 (CI: 2.17-9.33). Eight studies evaluated pregnancy rate after ART, showing a significant OR of 1.60 (CI: 1.08-2.37). Sub-dividing studies according to the FSH preparations (purified/recombinant), pregnancy rate improvement remained significant for each preparation. Eleven studies considered sperm quality after FSH treatment, finding a significant improvement of sperm concentration (2.66×10(6)/ml, CI: 0.47-4.84), but not of concentration of sperm with progressive motility (1.22×10(6)/ml, CI: -0.07 to 2.52). Three trials evaluated testicular volume, showing a non-significant increase in men treated (1.35 ml, CI: -0.44 to 3.14). CONCLUSION: The results of controlled clinical trials available in the literature indicate an improvement of pregnancy rate after FSH administration to the male partner of infertile couples, both spontaneously and after ART. However, the heterogeneity of studies, the high risk of bias and the lack of precise criteria to guide FSH administration limit the strength of these results. Future studies should be designed to identify the markers of FSH response which are helpful in the decision-making process. Meanwhile, the use of FSH in the treatment of male infertility should be cautious.

Lesions of epinephrine neurons in the rostral ventrolateral medulla abolish the vasodepressor components of baroreflex and cardiopulmonary reflex.

Epinephrine-containing neurons of the rostral ventrolateral medulla (RVL) (the C1 group of Hökfelt) in the rat are primarily unilaterally innervated by neurons in the nucleus tractus solitarius (NTS) and in turn project to autonomic spinal neurons. In this study, we investigated whether the C1 area of the RVL mediates the vasodepressor responses (VDR) induced by either electrical stimulation of the vagus nerve or carotid sinus stretch. In all experiments, C1 neurons were localized immunocytochemically with antibodies to phenylethanolamine N-methyltransferase (PNMT). Bilateral lesions of the C1 area decreased arterial pressure (AP) and heart rate (HR) to spinal cord transection levels and blocked the VDR induced by vagal stimulation and carotid sinus stretch. Combined lesions of the contralateral NTS and C1 area ipsilateral to the stimulated vagus nerve maintained AP and HR at normal levels, and totally blocked the VDR to vagal stimulation and carotid sinus stretch. Since projections from the vagus nerve to NTS are bilateral and those from NTS to C1 unilateral, the combined contralateral NTS/ipsilateral C1 lesions isolated and interrupted the ipsilateral NTS-C1 pathway and, therefore, blocked the baroreceptor reflex. The results are consistent with the hypothesis that neurons in the NTS synapsing in or projecting through the C1 area mediate the baro- and cardiopulmonary mechanoreceptor reflex.

Brain stem catecholamine mechanisms in tonic and reflex control of blood pressure.

Neurons of the lower brain stem maintain resting levels of arterial pressure (AP), mediate reflex responses from cardiopulmonary receptors, and are an important site of the hypotensive actions of alpha 2-adrenergic agonists. Details of the pathways and transmitters that mediate tonic and reflex control of AP are emerging. Afferent fibers of cardiopulmonary receptors in the ninth and tenth nerves terminate bilaterally in the nucleus of the tractus solitarius (NTS). Although some neurons contain substance P, the primary neurotransmitter appears to be the excitatory amino acid L-glutamate (L-glu). Neurons in rostral ventrolateral medulla, which most probably comprise the C1 group of epinephrine neurons, are also critical in AP control. C1 neurons project to innervate cholinergic preganglionic sympathetic neurons in the spinal cord. Stimulation of the C1 area electrically or with L-glu increases AP, while lesions or local injection of the inhibitory amino acid gamma-aminobutyric acid (GABA) lowers AP to levels comparable to spinal cord transection. Lesions of C1 neurons or their pathways abolish vasodepressor reflexes from baroreceptors and vagal afferents. In contrast, noradrenergic neurons of the caudal ventrolateral medulla, the A1 group, project rostrally to innervate, in part, vasopressin neurons of the hypothalamus. Stimulation of A1 neurons lowers AP, while lesions or GABA elevates it. We propose that C1 neurons comprise the so-called tonic vasomotor center of the brain stem and also mediate, via a projection from the NTS, the vasodepressor limb of baroreflexes. The NTS-C1 projection may be GABAergic.

A role of insular cortex in cardiovascular function.

We sought to determine whether the insular cortex contributes to the regulation of arterial blood pressure (AP). Responses to electrical and chemical stimulation of the cortex were studied in the anesthetized, paralyzed, and artificially ventilated Sprague-Dawley rat. The insular cortex was initially defined, anatomically, by the distributions of retrogradely labeled perikarya following injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus tractus solitarii (NTS). Injections of WGA-HRP into the insular cortex anterogradely labeled terminals in cardiopulmonary and other divisions of the NTS and confirmed projections revealed by retrograde tracing experiments. Electrical stimulation of the insular cortex elicited elevations of AP (less than or equal to 50 mm Hg) and cardioacceleration (less than or equal to 40 bpm). The locations of the most active pressor sites corresponded closely to the locations of retrogradely labeled cells in layer V of granular and posterior agranular areas of the insular cortex (areas 14 and 13) and the extreme capsule. Maximal pressor responses were obtained at a stimulus intensity of three to five times threshold current of 20-30 microA. Responses elicited mostly with higher-threshold currents were also mapped in areas 2a and 5lb and the claustrum and within the corpus callosum. Unilateral injections into the insular pressor area of the excitatory amino acid monosodium glutamate (L-Glu; 0.05 nmol to 10 nmol) or the rigid structural analogue of L-Glu, kainic acid (KA) (0.4 nmol) (which specifically excite perikarya), caused topographically specific elevations in AP and tachycardia. During the course of the anatomical transport studies, new findings were obtained on the organization and characteristics of the cortical innervation of the NTS and the nucleus reticularis parvocellularis. Topographic relationships between the cortex and the NTS were organized in a more complex manner than previously thought. Cells projecting to caudal cardiopulmonary segments of the NTS were fewer and generally located ventrally and caudally and in a more restricted area than cells projecting rostrally or to the parvicellular reticular formation. Anterograde transport data revealed new presumptive terminal fields in dorsolateral, ventral, periventricular, and commissural regions of the NTS, including an area overlapping the terminal field of the aortic baroreceptor nerve. We conclude that neurons within an area of the insular cortex projecting to multiple brainstem autonomic nuclei, including a region of the NTS innervated by baroreceptor afferents, increase arterial blood pressure and heart rate.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute sinoaortic denervation induces pre- and postsynaptic alpha-adrenergic subsensitivity in mesenteric arteries in the rat.

Bilateral sinoaortic denervation induced a rapid increase in blood pressure and heart rate in the rat. Four hours after sinoaortic denervation, the dose-response curve to l-norepinephrine (NE) in perfused mesenteric arteries in vitro was shifted to the right and the maximal response reduced, compared with sham-operated controls. These changes were abolished in the presence of cocaine (10 micrograms/ml). Four hours after sinoaortic denervation, the dose-response curves to methoxamine and serotonin, but not to adenosine-5'-triphosphate (ATP), were significantly reduced and the muscular response induced by barium chloride was not modified. Vascular responses to stimulation with different frequencies of the sympathetic nerve were reduced 4 hr after sinoaortic denervation. These changes in both the responses to l-NE and to nerve stimulation were reversed 24 hr after sinoaortic denervation. In the same preparation, 4 hr after sinoaortic denervation, the fractional release per pulse of [3H]-NE was significantly enhanced at 10 and 20 Hz frequencies of stimulation, whereas the blockade of the alpha-presynaptic receptors with phentolamine (3.1 microM) did not increase the release of [3H]-NE induced by nerve stimulation, as was observed in sham-operated controls. The results are compatible with the hypothesis that 4 hr after sinoaortic denervation, the pre- and postsynaptic alpha-adrenoceptors are desensitized. These changes could affect arterial sympathetic neurotransmission; the increase in blood pressure observed following sinoaortic denervation could therefore not be explained only by an increase in sympathetic tone.

Contributions of arginine vasopressin and the sympathetic nervous system to fulminating hypertension after destruction of neurons of caudal ventrolateral medulla in the rat.

We sought to determine the role in control of arterial pressure (AP) of the caudal ventrolateral medulla (CVL), a region containing noradrenergic neurons of the A1 group. Electrical stimulation of the A1 area in anaesthetized rats elicits a fall of AP and heart rate at low, but not high, stimulus frequencies. Electrolytic lesions of the A1 area produce fulminating hypertension and a 13-fold increase in plasma arginine vasopressin (AVP). A1-hypertension is attenuated by treatment with an AVP antagonist and is also diminished in rats of the Brattleboro strain. After AVP blockade, the residual hypertension is abolished by treatment with ganglionic or alpha adrenergic blockers, but not by adrenalectomy. We conclude that the region of the CVL containing the A1 noradrenergic cells tonically inhibits the discharge of sympathetic nerve activity as well as the release of AVP from the pituitary.

Effects of cocaine or denervation on responses of isolated strips of cat spleen to (-)-noradrenaline and (-)-isoprenaline.

1. A study has been made of the effects of cocaine and sympathetic denervation on responses of the cat spleen to (-)-noradrenaline and (-)-isoprenaline.2. Responses of isolated strips of spleen capsule to (-)-noradrenaline or to (-)-isoprenaline were not affected by reserpine-pretreatment.3. In adult cats, cocaine (1 and 10 mug/ml) or denervation produced a shift to the left of dose-response curves to (-)-noradrenaline, whereas they failed to modify dose-response curves to (-)-isoprenaline.4. There was an increase in the maximum development of tension to (-)-noradrenaline after denervation or in the presence of cocaine (10 mug/ml). These procedures did not increase the maximal responses to (-)-isoprenaline.5. Cocaine (10 mug/ml) did not affect dose-response curves to (-)-noradrenaline or (-)-isoprenaline in the denervated spleen.6. In the kitten spleen, cocaine (1 and 10 mug/ml) produced a shift to the left of dose-response curves to (-)-noradrenaline both in untreated and in reserpine-pretreated tissues.7. There was a small shift to the left in dose-response curves to (-)-isoprenaline in the presence of cocaine in the untreated but not in the reserpine-pretreated kitten spleen.8. It is concluded that the potentiation of responses to (-)-noradrenaline in the cat's spleen is due to a prejunctional effect of cocaine.

Congenital estrogen deficiency: in search of the estrogen role in human male reproduction.

Recently, a remarkable progress has been made in our understanding about the role of sex steroids in male physiology. In this paper, we consider the clinical aspects of congenital estrogen deficiency - notably, estrogen resistance and aromatase deficiency - in men and we discuss both well-established and supposed estrogen roles in the human male reproductive function. These topics include the role of estrogens in the control of gonadotropin secretion, in male fertility determination and psychosexual behavior. Briefly, estrogens play a pivotal role in the control of serum gonadotropin concentrations in the human male. Furthermore, a possible role of estrogens on both human male fertility and sexuality has also been suggested by recent studies, even though the available data are far from being conclusive. Conversely, for what concern fertility and sexual behavior, a well-established effect of estrogens has been provided by recent studies on male rodents, which show impaired sexual behavior and fertility as a consequence of estrogen defect.

The effect of chronic treatment with GH on gonadal function in men with isolated GH deficiency.

Eleven adult males, previously submitted to neurosurgery because of a pituitary lesion (three with craniopharyngioma, three with clinically non-functioning adenoma and five with macroprolactinoma) were treated with recombinant GH for 12 months after the diagnosis of GH deficiency was made. Circulating FSH, LH, prolactin, testosterone, 17 beta-estradiol (E2), dehyroepiandrosterone (DHEA-S), androstenedione. 17-OH-progesterone (17OHP), IFG-I, and steroid hormone-binding protein (SHBG) levels were assayed before and after CG test at study entry and 6 and 12 months after GH treatment. A significant increase in plasma IGF-I levels was obtained after 6 and 12 months of GH treatment. In addition, CG-stimulated, but not baseline, testosterone levels showed a significant increase after 6 and 12 months of GH treatment when compared with study entry (9.6 +/- 0.5 and 9.9 +/- 0.5 vs 7.9 +/- 0.5 ng/ml; P < 0.05). Baseline, but not CG-stimulated, serum 17OHP levels were significantly increased only after 12 months of GH treatment (1.7 +/- 0.1 vs 1.4 +/- 0.1 ng/ml; P < 0.05). No significant difference was found as far as both basal and CG-stimulated E2, androstenedione, DHEA-S and SHBG were concerned. With regards to the semen analysis, only seminal plasma volume was significantly increased after 12 months of GH treatment (2.9 +/- 0.3 vs 1.7 +/- 0.3 ml; P < 0.05). No significant change in sperm count, motility and abnormal forms was observed. These data show that GH treatment displays a clear-cut effect upon Leydig cell function and increases the production of seminal plasma volume in fertile adult males with isolated GH deficiency.

The effects of testosterone replacement on nocturnal penile tumescence and rigidity and erectile response to visual erotic stimuli in hypogonadal men.

Nocturnal penile tumescence (NPT) and erectile response to visual erotic stimuli (VES) were measured, by means of a Rigiscan device, in nine hypogonadal men, and repeated after 3 months of androgen replacement. The same assessments were carried out once in 12 eugonadal controls. The number of satisfactory NPT responses, in terms of both circumference increase and rigidity, were less in the hypogonadal men than the controls and were significantly increased by androgen replacement, confirming the results of earlier studies. In terms of circumference increase, erectile response to VES did not differ between the hypogonadal men and the controls, and did not increase with androgen replacement. In terms of rigidity, the erectile response to VES did not differ between hypogonadal men and controls. However, in terms of both duration and maximum level of rigidity, there was a significant increase following androgen replacement in the hypogonadal men. These new findings, in relation to rigidity, require a modification of the earlier formulation, which saw NPT as androgen dependent and erectile response to VES as androgen independent. NPT, and possibly spontaneous erections at other times, clearly involve an androgen sensitive system. Erectile response to VES predominantly involves an androgen independent system but may also be influenced by androgen sensitive mechanisms.

The endocrine effects of visual erotic stimuli in normal men.

Endocrine responses to erotic stimulation in the laboratory were assessed in eight normal subjects. Each subject was tested on two occasions. On one occasion only neutral stimuli were involved. After 15 min baseline, 30 min of films were shown. For the erotic condition on the other occasion, two 10-min erotic films were interspersed with 10 min of neutral film. Fifteen-minute blood samples were taken from the start of each test and continued for 5 hr after the films. Plasma was assayed for testosterone, LH, prolactin, cortisol, ACTH and beta-endorphin. Urine was collected for 4 hr before and 4 hr after the films; this was assayed for adrenaline, noradrenaline and dopamine. Sexual arousal occurred in response to the erotic films in all subjects, as shown by erectile and subjective responses. There were no significant changes in hormone or catecholamine levels following either the erotic or the neutral stimuli, except for a rise in cortisol during the neutral but not the erotic film. These results indicate that in the laboratory, substantial sexual response can occur without accompanying endocrine or biochemical changes.

Ascending and descending convergent inputs to neurons in the nucleus parabrachialis of the rat: an intracellular study.

Responses of the nucleus parabrachialis neurons (PBN) to electrical stimulation of the lateral hypothalamus (HL), central nucleus of the amygdala (Ac), dorsolateral funicullus in the spinal cord (SC), mediocaudal nucleus tractus solitarius (NTS), and substantia nigra (SN) were investigated in anesthetized rats by intracellular recording technique. Convergent excitatory postsynaptic potentials (EPSPs) were evoked on 8 of 36 neurons tested by both HL and NTS stimulation. The EPSPs evoked by HL stimulation were characterized as monosynaptic in 4 neurons. The EPSPs evoked by SC stimulation were characterized as monosynaptic in 2 of 36 neurons, moreover, these neurons were also antidromically activated by HL stimulation. Stimulation of Ac evoked EPSPs on 10 of 36 cells tested; 8 demonstrated to be monosynaptic. In addition, IPSP evoked by SN stimulation and EPSP evoked by NTS stimulation converged on three neurons. The results indicate that ascending and descending inputs converge on lateral PBN neurons.

Rostral ventrolateral medulla descending neurons excited by nucleus tractus solitarii inputs.

Neurons in the rostral ventrolateral medulla (RVLM) were electrophysiologically characterized and anatomically identified using an intracellular recording technique in vivo. Of 49 neurons recorded, 7 were antidromically activated from the dorsolateral funiculus in the thoracic spinal cord, with axonal conduction velocities ranging from 16.6 to 55.0 m/s. The RVLM-spinal neurons were spontaneously active and non-bursting. Additionally, they demonstrated a flat post-R-wave histogram and a flat average of the neuronal membrane potential triggered by the pulsatile arterial pressure. Therefore their activity was not related to cardiac rhythm. Electrical stimulation of the nucleus tractus solitarii (NTS) at the level of the obex evoked monosynaptic excitatory postsynaptic potential (EPSP) on 3 RVLM-spinal neurons; median latency was 1.5 ms. The recorded neurons, intracellularly labeled with horseradish peroxidase (HRP) or biocytin, were located in the rostral pole of the RVLM, between 0.3 and 0.7 mm from the ventral medullary surface and in many cases close to the neurons containing phenylethanolamine-N-methyltransferase (PNMT). These findings are discussed in relation to the physiological role in cardiovascular and nociceptive functional regulation played by the neurons analyzed in this study.

Relationship of calbindin D-28k with afferent neurons to the rostral ventrolateral medulla in the rat.

The phenylethanolamine-N-methyltransferase (PNMT)-containing neurons in the rostral ventrolateral medulla (RVLM) (the C1 adrenergic group) have been implicated in the generation of the tonic sympathetic nerve activity. Using a double-labeling immunohistofluorescence technique, we found that 34.6 +/- 11.4% (mean +/- S.D.) of PNMT immunoreactive neurons in the RVLM were immunoreactive for Calbindin D-28k (CaBP), a Vitamin D-dependent calcium binding protein. Since CaBP is probably involved in regulating intracellular calcium concentrations in cells that are metabolically or electrically very active, our results suggest that at least some C1 adrenergic neurons (those containing calbindin) may have calcium mediated high metabolic or electrophysiologic activity that is associated with generating tonic nerve function. The RVLM has wide connections with many different nuclei in the brain which are known to contain clusters of neurons that express immunoreactivity to CaBP. In order to determine whether CaBP could be used as a molecular marker for projection neurons to the RVLM or to identify a subpopulation of projection neurons containing CaBP, we sought to determine the relationships between CaBP and the neurons that project to RVLM. Following injections of the retrograde tracer FluoroGold (FG) into the rat RVLM, sections containing retrogradely labeled neurons in (1) the nucleus tractus solitarii (NTS), (2) the contralateral RVLM, (3) the area postrema, (4) the mesencephalic central gray (mCG), (5) the lateral hypothalamus (LH), (6) the substantia innominata (SI), and (7) the paraventricular hypothalamic nucleus (PV) were tested for CaBP immunoreactivity. Although many retrogradely labeled neurons were found amidst many CaBP immunoreactive neurons in each of these nuclei, only a subpopulation of the retrogradely labeled neurons expressed CaBP immunoreactivity. The NTS demonstrated the higher proportion of double-labeled cells (mean 31.5 +/- 4.3%), whereas the lower proportion corresponded to the contralateral RVLM (mean 9.6 +/- 3.2%). On the other hand, both the retrogradely labeled neurons and the CaBP immunoreactive neurons in each of these nuclei were often found in regions containing a great number of adrenergic axons (i.e. immunoreactive for PNMT). Our results suggest that: (1) Two types of adrenergic RVLM neurons could be found, those containing CaBP and those lacking this calcium binding protein. (2) CaBP is not a common marker for the afferent neurons to the RVLM, but rather is found in selective subsets of them. (3) Both the non-CaBP projection neurons and the CaBP immunoreactive neurons in these nuclei may be innervated by adrenergic fibers.