We set out to determine the projections of the major immunohistochemically-defined populations of intrinsic cardiac neurons to different target tissues within the guinea-pig heart. Ultrastructural studies, and immunoreactivity to the neuronal marker, neuron-specific enolase, suggested that the number of axons of intrinsic neurons in most regions of the heart was low when compared with the populations of axons projecting from extrinsic sensory and sympathetic ganglia. Multiple-labelling immunofluorescence was used to demonstrate the terminals of the major populations of peptide-containing intrinsic neurons. The intrinsic nature of peptide-containing axons was confirmed by long-term organotypic culture of cardiac tissue, which resulted in degeneration of axons of extrinsic neurons. The relative density and peptide content of intrinsic axons throughout the heart was not consistent with the relative proportions of peptide-containing intracardiac nerve cell bodies observed previously. The most commonly-encountered axons contained immunoreactivity (IR) to vasoactive intestinal peptide (VIP) alone, although nerve cell bodies with VIP constituted less than 5% of the total population of intrinsic neurons. Populations of axons containing IR to somatostatin alone, somatostatin and substance P, neuropeptide Y (NPY) alone, somatostatin and NPY, or VIP and NPY, also were observed. Intrinsic axons containing substance P-IR were very rare, much more so than would be predicted from the peptide content of intrinsic nerve cell bodies. The regions of the heart with the most dense innervation by axons of intrinsic neurons were the cardiac valves, the atrio-ventricular node and the sino-atrial node, Each of these targets was innervated by several populations of peptide-containing axons. Thus, each population of peptide-containing intrinsic neurons projected to a variety of target tissues within the heart. One possible interpretation of these results is that immunohistochemically-distinct populations of intrinsic neurons belong to different functional classes of neurons (sensory neurons, interneurons, final motor neurons), each of which innervates many regions of the heart.

A strategy has been developed to identify and quantify the different neurochemical populations of myenteric neurons in the guinea-pig ileum using double-labelling fluorescence immunohistochemistry of whole-mount preparations. First, six histochemical markers were used to identify exclusive, non-overlapping populations of nerve cell bodies. They included immunoreactivity for the calcium binding proteins calbindin and calretinin, the neuropeptides vasoactive intestinal polypeptide, substance P and somatostatin, and the amine, 5-hydroxytryptamine. The sizes of these populations of neurons were established directly or indirectly in double-labelling experiments using a marker for all nerve cell bodies. Each of these exclusive populations was further subdivided into classes by other markers, including immunoreactivity for enkephalins and neurofilament protein triplet. The size of each class was then established directly or by calculation. These distinct, neurochemically-identified classes were related to other published work on the histochemistry, electrophysiology and retrograde labelling of enteric neurons and to the simple Dogiel morphological classification. A classification scheme, consistent with previous studies, is proposed. It includes 14 distinct classes of myenteric neurons and accounts for nearly all neurons in the myenteric plexus of the guinea-pig ileum.

1. Intracellular recordings were made from the parasympathetic ganglion cells that lie in the epicardium of the left atrium of guinea‐pig heart near the interatrial septum. 2. Three distinct types of neurone were identified on the basis of their electrophysiological properties. In one group of neurones, S cells, somatic action potentials were followed by brief after‐hyperpolarizations. In the other two sets of neurones, somatic action potentials were followed by prolonged after‐hyperpolarizations. The neurones with prominent after‐hyperpolarization were further subdivided: one group of neurones, P cells, showed inward rectification at membrane potentials near the resting membrane potential whilst neurones in the other group, SAH cells, did so only at more negative potentials. 3. In the group of neurones that displayed inward rectification at potentials near rest, rectification resulted from the activation of an inward current, which resembled the hyperpolarization‐activated inward current present in cardiac muscle pacemaker cells. 4. The three different types of neurone received different patterns of synaptic input. Each SAH cell received a synaptic excitatory connection from the vagus which in most cells released sufficient transmitter to initiate an action potential in that cell; several SAH cells also received a separate connection, which could be activated by local stimulation. Although most S cells failed to receive a synaptic input from the vagus, all of those tested received an excitatory synaptic input which could be activated by local stimulation. Virtually all P cells failed to receive a synaptic input from the vagus; in addition, local stimulation failed to initiate synaptic potentials in P cells. 5. When the structure of cardiac ganglion cells was determined, by loading the cells with either biocytin or neurobiotin, it was found that most cells lacked extensive dendritic processes. S cells were invariably monopolar, most P cells were dipolar or pseudodipolar, whereas many SAH cells were multipolar. 6. In many neurones an on‐going discharge of action potentials was detected in the absence of obvious stimulation. In S and SAH cells, the action potentials resulted from an on‐going discharge of excitatory synaptic potentials. However, when a spontaneous discharge of action potentials was detected in P cells a discharge of excitatory synaptic potentials was not detected. 7. The results are discussed in relation to the idea that the three different types of cell may have different functions and that some of the cells may be organized in such a way as to permit the local handling of neuronal information within the heart.

Recent studies of autonomic ganglia have shown that specific combinations of neuropeptides and other potential neurotransmitters distinguish different functional types of neurons. In the present paper the patterns of coexistence of neurochemicals in guinea-pig cardiac ganglion cells was examined, using multiple-labelling immunohistochemistry. Many neurons were found to contain somatostatin immunoreactivity with various combinations of immunoreactivity for dynorphin B, substance P, neuropeptide Y and nitric oxide synthase. There were several small populations of neurons without somatostatin immunoreactivity, which contained combinations of immunoreactivity for vasoactive intestinal peptide, neuropeptide Y, dynorphin B, substance P and nitric oxide synthase. Possible synaptic inputs to these populations of ganglion cells were identified using multiple-labelling immunohistochemistry combined with long-term organ culture. These experiments demonstrated that cardiac ganglia contain prominent pericellular baskets of varicose nerve terminals of sympathetic and sensory origin. In addition, populations of intrinsic intraganglionic nerve terminals were identified which were immunoreactive for vasoactive intestinal peptide, neuropeptide Y or both peptides. These terminals presumably originate from intrinsic neurons, with the same combinations of neuropeptides, located in other cardiac ganglia.These results have demonstrated that there are diverse populations of cardiac ganglion cells in the guinea-pig and that some of these neurons may act as interneurons within the intrinsic cardiac plexuses. Therefore it is highly likely that vagal transmission in the heart is modified by sympathetic, sensory and intrinsic neurons and that cardiac ganglia are complex integrators of convergent neuronal activity rather than simple relays.

Heart rate is regulated by the autonomic nervous system but little is known about the pattern of innervation of the pacemaker in the sinoatrial node, or the subpopulations of nerves involved. Therefore in this study the pacemaker was located using electrophysiological methods and the pattern of innervation established by cholinesterase staining. In subsequent experiments, subpopulations of sympathetic, sensory and parasympathetic nerves were identified. Sympathetic nerves were labelled by glyoxylic acid-induced catecholamine fluorescence or an antiserum raised against tyrosine hydroxylase (TH). These experiments showed that the entire sinoatrial node was densely innervated by sympathetic axons, the majority of which were immunoreactive for neuropeptide Y (NPY). There were a few axons which were only immunoreactive for TH. Sensory nerves which were immunoreactive for both substance P (SP) and calcitonin gene-related peptide (CGRP) were also found throughout the sinoatrial node. In the absence of a selective marker for parasympathetic neurons, hearts were extrinsically denervated by placing them in organotypic culture to allow degeneration of extrinsic axons. In this way intrinsic parasympathetic neurons could be characterised. These experiments revealed several distinct populations of parasympathetic nerves which innervated only a small, discrete part of the sinoatrial node. These populations were immunoreactive for NPY, somatostatin (SOM) or vasoactive intestinal peptide (VIP) alone, or SOM combined with NPY, SOM with dynorphin B, and SOM with SP. These results highlight a remarkable difference in the pattern of innervation of the sinoatrial node by the sympathetic and parasympathetic nervous systems. Furthermore the presence of several distinct populations of autonomic cardiac neurons indicates a further complexity in neuronal regulation of heart rate.

Motor neurons that innervate the longitudinal muscle of the guinea pig ileum were identified by retrograde transport from the longitudinal muscle plexus in organotypic culture. Motor neurons had short projections, less than 3.5 mm long, and never had Dogiel type II morphology; most labeled neurons had morphological characteristics of Dogiel type I neurons. Immunoreactivity for choline acetyltransferase was present in 97% of retrogradely labeled nerve cell bodies, reflecting the dominant cholinergic input to the longitudinal muscle layer. Substance P immunoreactivity was present in 48% of motor neurons, indicating that it or a similar tachykinin that mediates noncholinergic excitatory transmission is likely to be released by a subset of cholinergic motor neurons. This strongly suggests that the difference in frequency dependence of substance P and acetylcholine release is attributable to different release mechanisms rather than to activation of separate populations of motor neurons. Immunoreactivity for the calcium-binding protein calretinin was present in 87% of longitudinal muscle motor neurons. The neurochemical coding of longitudinal muscle motor neurons indicated that they constitute about one quarter of all myenteric neurons and are distinct from circular muscle motor neurons.

Double-labelling immunohistochemistry and retrograde transport of the carbocyanine dye, DiI, were used to establish the pathways of submucous neurons to the mucosa of the guinea-pig small intestine. Following the application of DiI to a villus, DiI-labelled nerve cell bodies were found in the submucous plexus up to 8.3 mm circumferentially and 3.8 mm longitudinally. The size of each of the four characterised classes of submucous neurons was determined and their distributions and projections mapped. Cells characterised by vasoactive intestinal polypeptide immunoreactivity accounted for 52% of DiI-labelled cells and had the longest projections. Cells characterised by neuropeptide Y (19%) or by calretinin immunoreactivity (13% of all DiI-labelled neurons) had relatively short projections and cells with substance P immunoreactivity (20%) had intermediate lengths of projection. When DiI was applied directly to the submucous plexus, filled neurons of all classes had significantly shorter projections, indicating that they must run for considerable distances in other pathways to the mucosa, probably via the non-ganglionated plexus. On average, each villus is innervated by at least 70 submucous neurons. From quantitative estimates there are 9 submucous neurons per villus. Thus, each submucous neuron is likely to supply about 8 villi. This demonstrates a high degree of convergence and divergence in the innervation of the mucosa.

Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26 +/- 1% of myenteric neurones and 12 +/- 3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.

Motor neurons which innervate the circular muscle layer of the guinea-pig small intestine were retrogradely labelled, in vitro, with the carbocyanine dye, DiI, applied to the deep muscular plexus. By combining retrograde tracing and immunohistochemistry, the chemical coding of motor neurons was investigated. Five classes of neuron could be distinguished on the basis of the co-localization of immunoreactivity for the different antigens; the five classes were also characterized by different lengths and polarities of their axonal projections and by their cell body shapes. Two classes with local or orally directed axons were immunoreactive for choline acetyltransferase and substance P and are likely to be cholinergic excitatory motor neurons. Two other classes had anally directed axons; they were immunoreactive for vasoactive intestinal polypeptide and are likely to be inhibitory motor neurons. A small proportion of neurons with short projections to the circular muscle were immunoreactive for neither substance P nor for vasoactive intestinal polypeptide, but are likely to be cholinergic. The morphological and histochemical identification of excitatory and inhibitory motor neurons provides a neuroanatomical basis for the final motor pathways involved in the polarized reflex motor activity of the gut.

SUMMARY LH pulse frequency and amplitude vary significantly during the menstrual cycle; however, it is not clear what significance the secretory pattern has for the ovary. We have developed an in‐vitro perifusion system in which luteinized human granulosa cells (GC) can be exposed to various patterns of gonadotrophin stimulation. GC were recovered following follicle aspiration for in‐vitro fertilization, grown on Cytodex‐3 for 6 days, and then perifused with medium containing LH (or hCG), delivered with differing pulse frequencies and amplitudes. When pulses of LH were applied to the cells, progesterone secretion rose initially and then fell to the baseline as the LH concentration declined. Pulsatile administration of LH, over a period of 10 h, stimulated progesterone secretion more efficiently than did continuous LH. Finally, delivery to the cells of pulses of hCG, a ligand known to bind to the LH receptor but with binding characteristics distinct from those of LH, failed to elicit pulses of progesterone.

Guinea-pig ileum was dissected and the mucosa, submucosa and external musculature extracted with aqueous acetic acid for measurement of four prodynorphin-derived peptides, namely dynorphin A 1–8, dynorphin A 1–17, dynorphin B, and α-neoendorphin. The peptide-like immunoreactive material extracted from the external musculature was characterized by multi-dimensional chromatographic analysis and compared to synthetic porcine standards. The chromatographic methods utilized were: reversed-phase high performance liquid chromatography (RP-HPLC), using two different eluants; cation exchange high performance liquid chromatography (CE-HPLC) and gel filtration chromatography. The dynorphin A 1–8-like immunoreactive material was homogeneous and coeluted with the standard in all chromatographic modes. The dynorphin A 1–17-like and dynorphin B-like immunoreactive material was heterogeneous but showed a peak that coeluted with synthetic standard in all chromatographic modes. The α-neoendorphin-like immunoreactive material also appeared to be heterogeneous with the major component on CE-HPLC coeluting with the synthetic peptide standard while the major component on RP-HPLC eluted differently. It was concluded that the guinea-pig ileum contains immunoreactivity for peptides derived from all coding regions of the prodynorphin gene and that these peptides may be present in multiple immunoreactive forms.

SUMMARY Progesterone is known to exert a biphasic feedback effect on luteinizing hormone (LH) secretion in animals and it has been demonstrated that this effect is dependent upon both duration of exposure to progesterone and the dose administered. In this paper we sought to determine whether a similar biphasic effect exists in humans. The pattern of LH secretion was assessed in six healthy oestrogen treated post‐menopausal women before and after they were given progesterone (50 mg/day) for 1 and 7 days. Progesterone treatment for 1 day resulted in a significant elevation in the basal serum LH concentration and in individual LH pulse amplitude with no change in LH pulse frequency. In contrast, progesterone treatment for 7 days increased LH pulse amplitude with no change in basal serum LH concentrations and a significant reduction in LH pulse frequency. We concluded that firstly, progesterone does exert a biphasic feedback effect on LH secretion and that the nature of this effect is determined by the duration of exposure to the progesterone stimulus. Secondly, as LH pulsatility has been shown to be an accurate indicator of GnRH pulsatility, that the reduction in LH pulse frequency after a long exposure to progesterone is due to a hypothalamic effect of progesterone whereas the positive feedback effect may be the result of a pituitary or hypothalamic action.

We assessed the frequency of luteinizing hormone (LH) pulsatility (reflecting the activity of gonadotropin-releasing hormone [GnRH] neurons in the hypothalamus) in six women during the periovulatory LH surge, in five women during the early follicular phase, and in seven women in the midfollicular phase (MFP) (calculated as being 3 to 8 days before the LH surge). Collection of blood at 5-minute, versus 15-minute, intervals allowed detection of a larger number of LH pulses in both the MFP (16, versus 27) and periovulatory phase (POP) (11, versus 22) groups of women, but it made no difference in the early follicular phase (EFP) (10 pulses with both methods). During the EFP, the mean number of LH pulses per 4 hours (detected by 5-minute sampling) was 2.0 +/- 0.7 (+/- standard deviation [SD]), and the mean LH amplitude (+/- SD) was 1.3 +/- 0.4 IU/l. There was a significant increase in the number of pulses in the MFP group (3.9 +/- 1.3 pulses/4 hours; P less than 0.05) but no significant change in pulse amplitude (1.1 +/- 0.1 IU/l). During the POP, the mean pulse amplitude was increased (8.5 +/- 1.4 IU/l; P less than 0.001), compared with the MFP and EFP groups, but the mean pulse frequency (3.7 +/- 1.2 pulses/4 hours) was not significantly different from the MFP frequency. We conclude that an acceleration of LH pulsatility occurs several days before the LH surge and does not change thereafter. However, there is an increase in LH pulse amplitude during the LH surge; we attribute this to the increase in pituitary sensitivity at this time.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was designed to evaluate the role of luteinizing hormone (LH) and prolactin (PRL) in regulating pulsatile progesterone secretion in the human. This was done first by correlating the frequency of progesterone, LH and PRL pulses during the mid-luteal phase of normal cycles. Second, by increasing the frequency of LH pulses with naloxone and GnRH injections and examining the impact on progesterone pulse frequency. Third, by abolishing PRL pulsatility with metoclopramide and looking at the effect on progesterone pulsatility. Nine normal subjects in the mid-luteal phase (4-10 days after the initial postovulatory rise in progesterone) were studied for 8 h with blood samples taken at 15 min intervals. Each sample was assayed for progesterone, LH and PRL and the pulse frequency (number of pulses in 8 h) determined for each hormone. The mean pulse frequencies were 2.3 (s.e.m. = 0.4) for progesterone, 1.3 (s.e.m. = 0.4) for LH, and 2.1 (s.e.m. = 0.3) for PRL. Cross-correlation analysis showed that there was no significant synchrony between pulses of progesterone and pulses of LH and PRL. When naloxone was given to six normal subjects in the mid-luteal phase, the mean LH pulse frequency (number of pulses in 6 h) was increased from 2.2 (s.e.m. = 0.3) during a saline infusion to 3.2 (s.e.m. = 0.5) during the naloxone infusion (P less than 0.05). However, the mean pulse frequency for progesterone remained unchanged during the saline and naloxone studies. There was no significant difference between the mean serum LH in the saline and naloxone groups, and the mean serum progesterone concentration was not significantly altered.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY This study was designed to assess the effect of an altered level of serum oestrogen and progesterone on the prolactin (PRL) response to gonadotrophin releasing hormone (GnRH). Six normal women were studied in the early follicular phase and the mid‐luteal phase of one cycle and five menopausal women were studied before and after treatment with progesterone. Blood samples were collected at 15 min intervals for 6 h after a basal collection period of 30 min. Intravenous boluses of GnRH (1 μg, 10 μg and 50 μg) were given at 0, 2 and 4 h. Basal samples were assayed for 17β‐oestradiol (E 2 ), oestrone (E 1 ) and progesterone (P); LH, FSH and PRL were measured in all samples. Serum PRL was significantly elevated in all groups after 10 μg of GnRH with maximum increments (±SEM) ranging from 3.9±1.3 μg/l in early follicular phase women to 14.7 ± 4.7 μg/l in progesterone‐treated menopausal women. The PRL response to GnRH was significantly greater in the luteal phase and in menopausal women compared to early follicular phase women. There was a significant correlation between the maximum PRL response and the maximum LH response to GnRH in all the women studied ( r = 0.7; P < 0.01). A significant correlation was also found between the maximum PRL response and the basal serum oestrogen concentration in the normal cycling women ( r = 0.8; P < 0.01), but not when the menopausal women were included in the analysis. Administration of progesterone to menopausal women did not significantly affect the PRL response to GnRH and no significant correlation was found between the basal serum progesterone and the PRL response to GnRH. We conclude that GnRH stimulates the secretion of PRL in normal, cycling and in menopausal women to a degree which is more closely correlated with the LH response than with the circulating oestrogen or progesterone concentration. This is consistent with the hypothesis that GnRH exerts an indirect effect on the lactotrophe by stimulating the prodution of a paracrine factor from the gonadotrophe. We suggest that the action of GnRH on the lactotrophe may be physiological in view of its reported action in synchronizing the secretion of LH and PRL and in maintaining the acute releasable pool of PRL in the lactotrophe.

SUMMARYIt is well‐established that the frequency of LH pulses varies during the normal menstrual cycle with a significant reduction in frequency in the luteal phase. Previous studies have indicated that both progesterone and opioids are able to reduce the frequency of LH pulses and in this study we sought to clarify the possible interaction between progesterone, endogenous opioids and GnRH neurons. Sixteen normal women in the mid‐follicular phase (days 8–12) were randomly allocated to a control or treatment group and LH pulsatility assessed on one or two occasions by taking blood samples at 15 min intervals over 8 h. For the control women, LH pulsatility was assessed on one occasion during a saline infusion. The treated women received progesterone (50–100 mg/d for 7 d) at the end of which LH pulsatility was assessed before and after a naloxone infusion (2 mg/h for 8 h). Mean±SEM LH pulse frequency in the control women was 4.9 ± 0.5 pulses/8 h which was significantly decreased to 3.0 ± 0.3 pulses/8 h (P < 0.01) in the progesterone treated women but not different from 5.5 ± 0.3 pulses/8 h in those also treated with naloxone. Mean ± SEM LH pulse amplitude in the control women was 2.3 ± 0.3 IU/l, which was significantly increased to 4.8 ± 0.7 IU/l (P < 005) in the progesterone treated group, and to 3.7 ± 0.4 IU/l (P < 005) in the progesterone‐treated women after naloxone. We conclude that progesterone slows the frequency of LH pulsatility by increasing endogenous opioid activity in the hypothalamus which may in turn inhibit the firing rate of the GnRH neurons.

Nine normal cycling subjects were monitored for mid-cycle LH surge and a subsequent rise in serum progesterone levels. Frequent samples were then taken for progesterone measurement during 8 h sessions in the early, mid and late luteal phases. These data showed: that progesterone secretion is pulsatile throughout the human luteal phase, with maximum frequency in the mid-luteal phase; that during the mid-luteal phase most subjects had progesterone levels both above and below currently accepted ovulatory thresholds; the use of a single measurement of progesterone in the mid-luteal phase is not always a reliable indicator of ovulation; a threshold greater than 20 nmol/l may yield an unacceptable number of false negative results.