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Gonadotropin-releasing hormone (GnRH) neuron activity and GnRH secretion are essential for fertility in mammals. Here, I review findings from mouse studies on the direct modulation of GnRH neuron activity and GnRH secretion by non-peptide neurotransmitters (GABA, glutamate, dopamine, serotonin, norepinephrine, epinephrine, histamine, ATP, adenosine, and acetylcholine), gasotransmitters (nitric oxide and carbon monoxide), and gliotransmitters (prostaglandin E2 and possibly GABA, glutamate, and ATP). These neurotransmitters, gasotransmitters, and gliotransmitters have been shown to directly modulate activity and/or GnRH secretion in GnRH neurons in vivo or ex vivo (brain slices), from postnatal through adult mice, or in embryonic or immortalized mouse GnRH neurons. However, except for GABA, nitric oxide, and prostaglandin E2, which appear to be essential for normal GnRH neuron activity, GnRH secretion, and fertility in males and/or females, the biological significance of their direct modulation of GnRH neuron activity and/or GnRH secretion in the central regulation of reproduction remains largely unknown and requires further exploration.

Based on pharmacological studies, corticotropin-releasing hormone (CRH) and its receptors play a leading role in the inhibition of the hypothalamic-pituitary-gonadal (HPG) axis during acute stress. To further study the effects of CRH receptor signaling on the HPG axis, we generated and/or employed male mice lacking CRH receptor type 1 (CRHR1) or type 2 (CRHR2) in gonadotropin-releasing hormone neurons, GABAergic neurons, or in all central neurons and glia. The deletion of CRHRs revealed a preserved decrease of plasma luteinizing hormone (LH) in response to either psychophysical or immunological stress. However, under basal conditions, central infusion of CRH into mice lacking CRHR1 in all central neurons and glia, or application of CRH to pituitary cultures from mice lacking CRHR2, failed to suppress LH release, unlike in controls. Our results, taken together with those of the earlier pharmacological studies, suggest that inhibition of the male HPG axis during acute stress is mediated by other factors along with CRH, and that CRH suppresses the HPG axis at the central and pituitary levels via CRHR1 and CRHR2, respectively.

Leptin and melanocortin signaling control ingestive behavior, energy balance, and substrate utilization, but only leptin signaling defects cause hypothalamic hypogonadism and infertility. Although GnRH neurons do not express leptin receptors, leptin influences GnRH neuron activity via regulation of immediate downstream mediators including the neuropeptides neuropeptide Y and the melanocortin agonist and antagonist, α-MSH, agouti-related peptide, respectively. Here we show that modulation of melanocortin signaling in female db/db mice through ablation of agouti-related peptide, or heterozygosity of melanocortin 4 receptor, restores the timing of pubertal onset, fertility, and lactation. Additionally, melanocortin 4 receptor activation increases action potential firing and induces c-Fos expression in GnRH neurons, providing further evidence that melanocortin signaling influences GnRH neuron activity. These studies thus establish melanocortin signaling as an important component in the leptin-mediated regulation of GnRH neuron activity, initiation of puberty and fertility.

Kisspeptin plays an important role in puberty and subsequent fertility by activating its receptor, G-protein-coupled receptor 54 (GPR54), and increasing cytoplasmic free Ca2+ concentration ([Ca2+]i) and gonadotropin-releasing hormone (GnRH) secretion in GnRH neurons. Yet the mechanism by which kisspeptin increases [Ca2+]i in GnRH neurons remains to be fully elucidated. In other neurons, voltage-gated Ca2+ channel (VGCC) activity has been shown to be inversely related to [Ca2+]i. We used whole-cell patch-clamp recording to examine the effects of kisspeptin-10 (KP-10) on VGCC activity evoked by step depolarizations in GnRH neurons in brain slices from pubertal male GnRH-green fluorescent protein transgenic mice. Prolonged (>30 s) KP-10 application inhibited Ca2+ currents. The GPR54 antagonist peptide 234, chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid, substitution of Ba2+ for Ca2+, the calmodulin antagonists calmidazolium and trifluoperazine, the phospholipase C inhibitor edelfosine, the canonical transient receptor potential (TRPC) channel and inositol 1,4,5-trisphosphate receptor (IP3R) antagonist 2-APB, the TRPC channel antagonist BTP2 and the endoplasmic reticulum Ca2+-ATPase blocker cyclopiazonic acid each prevented inhibition. The IP3R antagonists caffeine (10 µM), heparin and intracellular 2-APB prevented inhibition to a lesser extent. The ryanodine receptor (RyR) antagonists ryanodine and dantrolene prevented inhibition, and the RyR agonist caffeine (30 mM) mimicked the effects of KP-10 on Ca2+ currents. Our results suggest that kisspeptin induces Ca2+ influx through TRPC channels and Ca2+ release via IP3Rs and RyRs, and that this is followed by Ca2+/CaM-dependent inhibition of VGCCs.

The pubertal increase in GnRH secretion resulting in sexual maturation and reproductive competence is a complex process involving kisspeptin stimulation of GnRH neurons and requiring Ca(2+) and possibly other intracellular messengers. To determine whether the expression of Ca(2+) channels, or small-conductance Ca(2+)-activated K(+) (SK) channels, whose activity reflects cytoplasmic free Ca(2+) concentration, changes at puberty in GnRH neurons, Ca(2+) and SK currents in GnRH neurons were recorded in brain slices of juvenile [postnatal day (P) 10-21], pubertal (P28-P42), and adult (or =P56) male GnRH-green fluorescent protein transgenic mice using perforated-patch and whole-cell techniques. Ca(2+) currents were inhibited by the Ca(2+) channel blocker Cd(2+) and showed marked heterogeneity but were on average similar in juvenile, pubertal, and adult GnRH neurons. SK currents, which were inhibited by the SK channel blocker apamin and enhanced by the SK and intermediate-conductance Ca(2+)-activated K(+) channel activator 1-ethyl-2-benzimidazolinone, were also on average similar in juvenile, pubertal, and adult GnRH neurons. These findings suggest that whereas Ca(2+) and SK channels may participate in the pubertal increase in GnRH secretion and there may be changes in Ca(2+) or SK channel subtypes, overall Ca(2+) and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Hence, the expected increase in GnRH neuron cytoplasmic free Ca(2+) concentration required for increased GnRH secretion at puberty appears to be due to mechanisms other than altered Ca(2+) or SK channel expression, e.g. increased membrane depolarization and subsequent activation of preexisting Ca(2+) channels after increased excitatory synaptic input.

The roles of ionotropic glutamate receptors in mammalian reproduction are unknown. We therefore generated mice lacking a major subtype of (S)-α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptors or all N-methyl-d-aspartate (NMDA) receptors in GnRH neurons and other mainly limbic system neurons, primarily in hypothalamic and septal areas. Male mice without NMDA receptors in these neurons were not impaired in breeding and exhibited similar GnRH secretion as control littermates. However, male mice lacking GluR-B containing AMPA receptors in these neurons were poor breeders and severely impaired in reproductive behaviors such as aggression and mounting. Testis and sperm morphology, testis weight, and serum testosterone levels, as well as GnRH secretion, were unchanged. Contact with female cage bedding failed to elicit male sexual behavior in these mice, unlike in control male littermates. Their female counterparts had unchanged ovarian morphology, had bred successfully, and had normal litter sizes but exhibited pronounced impairments in maternal behaviors such as pup retrieval and maternal aggression. Our results suggest that NMDA receptors and GluR-B containing AMPA receptors are not essential for fertility, but that GluR-B containing AMPA receptors are essential for male and female reproduction-related behaviors, perhaps by mediating responses to pheromones or odorants.

Secretion of gonadotropin-releasing hormone (GnRH) at the median eminence is the essential activator of the reproductive axis. The mechanisms by which embryonic GnRH neurons migrate from the olfactory placode to the preoptic area and then elaborate neurites that course through the hypothalamus to terminate at the median eminence are largely unknown. We investigated the hypothesis that GnRH neurite outgrowth is promoted by brain-derived neurotrophic factor (BDNF) because GnRH neurites course through BDNF-rich areas of the forebrain during their development. Confocal microscopy revealed that most (86%) cultured embryonic GnRH cells tagged with a green fluorescent protein reporter were immunoreactive for TrkB. In primary cultures of E12.5 olfactory tissue, treatment with BDNF induced a dose-dependent increase in neurite outgrowth, but had no discernible effect on branching. BDNF induced phosphorylation of Ca(2+)/cAMP response element-binding protein (pCREB) in both GnRH and non-GnRH cells in these cultures. This was not associated with phosphorylation of ERK in GnRH-immunoreactive cells, though BDNF treatment did stimulate pERK in neighbouring non-GnRH cells. Promotion of neurite outgrowth is unlikely therefore to result from activation of the Ras-MAPK/ERK pathway. We conclude that the developing GnRH secretory system is directly sensitive to BDNF and that this polypeptide functions as a neurotrophic factor for GnRH neurons.

To examine the role of calcium signaling in the joint modulation of gonadotropin-releasing hormone (GnRH) secretion by γ-aminobutyric acid (GABA) and glutamate, cytoplasmic calcium ([Ca2+]

Glutamate and its receptors are present in the hypothalamus and have been proposed to participate in neuroendocrine regulation, including the control of GnRH secretion. To address the mechanism of glutamate action, we measured [Ca2+]i, inositol phosphate, and secretory responses to glutamate receptor subtype agonists and antagonists in the immortalized GT1-7 cell line of GnRH-secreting hypothalamic neurons. Glutamate, N-methyl-D-aspartate (NMDA), kainate, and trans-(±)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid increased GnRH secretion. In monolayer cultures of GT1-7 cells, L- but not D-glutamate induced a moderate, concentration-dependent rise in [Ca2+]i. The action of glutamate on [Ca2+]i was mimicked by NMDA, α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA), and kainate. Responses to NMDA were potentiated by the coagonist, glycine, and were inhibited by an antagonist of the glycine site on the NMDA receptor, 5,7-dichlorokynurenic acid (DCKA). NMDA-induced [Ca2+]i responses were also inhibited by Mg2+ and by the NMDA receptor antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), but not by the AMPA/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, responses to AMPA and kainate were inhibited by CNQX but not by Mg2+, DCKA, or MK-801. Responses to glutamate were more inhibited by MK-801 plus CNQX than by either antagonist alone. All [Ca2+]i responses were nearly abolished in Ca2+-free solution. None of the agonists stimulated inositol phosphate formation. Hence, the NMDA and AMPA/kainate subtypes, but not the metabotropic subtype, of glutamate receptor are expressed in GT1-7 cells. Activation of NMDA and AMPA/kainate receptors is followed by Ca2+ influx and subsequently by GnRH secretion.

The dependence of the carotid chemosensory response to hypoxia on metabolic substrate and the hypothesis that lactic acidosis is essential for O2 chemoreception were tested. Effects of 3 types of substrate (glucose, glutamate and a mixture of amino acids) on the response to hypoxia (perfusate flow interruption) were measured (n = 33 carotid bodies). The response to nicotine (n = 25) was used to determine whether these effects were exclusive to the hypoxic response. The cat carotid body was perfused and superfused in vitro with modified Tyrode solution (pO2400 Torr, pCO21 Torr, pH = 7.4) at 36 degrees C containing a given substrate for at least 15 min prior to flow interruption or nicotine injection. Without substrate, responses to flow interruption (n = 4) and nicotine (n = 2) were irreversibly depressed. With glucose, responses to flow interruption (n = 13) and nicotine (n = 8) increased in a concentration-dependent fashion. Glutamate (42 mM) alone (n = 11) or a mixture of amino acids (4.2 mM) plus 5.5 mM glucose (n = 12) substituted for 11 mM glucose (n = 10). Thus, glutamate (42 mM), or a mixture of amino acids (4.2 mM) or a high concentration of glucose (11 mM) can support chemosensory responses to flow interruption and nicotine. Since glutamate undergoes oxidative deamination to alpha-ketoglutarate without lactic acid production, O2 chemoreception does not depend on lactic acidosis.

To test the hypothesis that O2 chemoreception in the carotid body (CB) is mediated by cellular acidosis, we simultaneously measured responses of the chemosensory and intracellular pH (pHi) to agents that are known to change pHi and studied the effects of hypoxia and ischemia on these variables in the cat CB. The CB was perfused and superfused in vitro with a modified Tyrode's solution at 36.0 +/- 0.5 degrees C with or without CO2-HCO3- (pH 7.40) and equilibrated at a given PO2. Chemosensory discharges were recorded from the whole carotid sinus nerve. To measure pHi changes, the CB was loaded with the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and the fluorescence (excitation 420-490 nm, emission greater than 515 nm) was detected by an intensified charged coupled device camera with an epifluorescence macroscope. Boluses of Tyrode's solution (0.5 ml, free of CO2-HCO3-) containing sodium acetate or NH4Cl prolonged perfusion of acid Tyrode's solution (pH 7.20-6.50), and boluses of Tyrode's solution with CO2-HCO3- were used. A decrease of fluorescence indicated pHi turning acid, and an increase of fluorescence indicated a change in alkaline pHi. Chemosensory activity varied inversely with the fluorescence change after application of these agents. Interruption of perfusate flow or application of hypoxic perfusate resulted in large increases in chemosensory discharge without any change in the fluorescence. The results indicated that chemosensory responses to brief ischemia and hypoxia were not mediated by a fall of pHi of CB cells, whereas those to CO2 and extracellular acidity were associated with decreases in pHi.

The relationship between oxygen pressure (PO2) in the carotid body and carotid sinus nerve discharge was evaluated in the isolated perfused/superfused cat carotid body using the oxygen-dependent quenching of phosphorescence. Images of phosphorescence intensity arising from Pd-coproporphyrin within the microcirculation of the carotid body provided measurements of intravascular PO2. These measurements were substantiated by determining phosphorescence life-time. The carotid body was perfused in the isolated state via the common carotid artery with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Tyrode solution, pH 7.4, at a constant pressure of 80 mmHg. Superfusion was maintained with similar media equilibrated with 100% argon. PO2 in the exchange vessels was markedly less than that in the perfusate entering the carotid artery, 23 +/- 3 and 45 +/- 3 Torr for normoxic (111 +/- 15 Torr) and hyperoxic (345 +/- 72 Torr) perfusates, respectively. Chemosensory discharge rose slowly in response to a brief interruption of perfusate flow as PO2 steadily declined from either of these capillary PO2 values to approximately 10 Torr. Between approximately 10 and 3 Torr, chemosensory discharge increased strikingly, concomitant with an enhanced rate of oxygen disappearance, from -36 +/- 4 to -69 +/- 13 (92% change) and -28 +/- 3 to -48 +/- 3 (71% change) Torr/s for normoxic and hyperoxic perfusates, respectively. As PO2 fell below approximately 3 Torr, oxygen disappearance slowed and neural activity decayed. Thus the relationships between microvascular PO2 and chemosensory discharge and between oxygen disappearance and neural discharge suggest that oxygen metabolism in the carotid body determines the expression of oxygen chemoreception.

The purpose of the present study was to determine whether the septo-preoptico-tuberoinfundibular gonadotropin-releasing hormone (GnRH) pathway comes in close juxtaposition with tyrosine hydroxylase immunoreactive (TH-IR) neurons in the arcuate nucleus of female mice. Immunohistochemical staining with a TH monoclonal antibody coupled with confocal microscopy was employed on vibratome-cut brain sections of female GnRH-green fluorescent protein (GFP) transgenic mice to evaluate possible appositions between GnRH and tuberoinfundibular dopaminergic (TIDA) neurons. TH-IR neurons of the arcuate nucleus received GnRH neuronal appositions in adult female mice at proestrus and estrus stages. In contrast, no GnRH appositions were observed in adult females at diestrus. Subsequently, double immunohistochemical staining for TH and estrogen receptor-α (ERα) was performed to examine the role of estradiol on this relationship. We found that most TH-IR neurons contacted by GnRH fibers were immunoreactive for ERα. Our observations suggest that GnRH neurons communicate directly with TIDA neurons in the adult female. Furthermore, ERα activation in TIDA neurons may be involved in the formation of connections between GnRH neurons and TIDA neurons.

Mice were generated expressing green fluorescent protein (GFP) under the control of the gonadotropin-releasing hormone (GnRH) promoter. Green fluorescence was observed in, and restricted to, GnRH-immunopositive neuronal somata in the olfactory bulb, ganglion terminale, septal nuclei, diagonal band of Broca (DBB), preoptic area (POA), and caudal hypothalamus, as well as GnRH neuronal dendrites and axons, including axon terminals in the median eminence and organum vasculosum of the lamina terminalis (OVLT). Whole-cell recordings from GFP-expressing GnRH neurons in the OVLT–POA–DBB region revealed a firing pattern among GFP-expressing GnRH neurons distinct from that of nonfluorescent neurons. Nucleated patches of GFP-expressing GnRH neurons exhibited pronounced responses to fast application of GABA and smaller responses tol-glutamate and AMPA. One-fifth of the nucleated patches responded to NMDA. The GABA-A, AMPA, and NMDA receptor channels on GnRH neurons mediating these responses may play a role in the modulation of GnRH secretory oscillations.

The pulsatile pattern of gonadotropin-releasing hormone (GnRH) release from the hypothalamus is driven by a functionally interconnected and synchronized network of GnRH neurons termed the GnRH pulse generator. Several recent observations have revealed that immortalized GnRH neurons can generate an episodic pattern of GnRH release when cultured in the absence of other cell types. The in vitro operation of the pulse generator depends on the development of synaptic contacts among GnRH neurons, the electrical properties of individual GnRH neurons, and the GnRH-induced modulation of its secretory mechanism. The expression o f several other receptors by GnRH neurons provides the means for integrated regulation of pulse generator activity from without the network by agonists including glutamate, GABA, endothelin, and catecholamines.

Publisher Summary Mammalian gonadotropin-releasing hormone (GnRH)-producing cells are located within the preoptic area or the mediobasal hypothalamus, depending on the species. In contrast to other hypothalamic neurons, GnRH cells do not form a clearly defined nucleus within which the cells are closely adjacent and tightly connected. Despite their scattered locations, GnRH neurons are interconnected to form a network termed the GnRH pulse generator that is responsible for the synchronized pattern of pulsatile GnRH release into the portal vessels in vivo as well as from hypothalamic tissue in vitro . An increase in intracellular calcium concentration ([Ca 2+ ] i ) is required for the release of neurotransmitters from presynaptic terminals in many neurons. Thus, it is reasonable to postulate that the pulsatile nature of basal GnRH release as well as agonist-mediated modulation of basal GnRH release is associated with changes in [Ca 2+ ] i .

The mechanism(s) of oxygen chemoreception by the carotid body is not understood. One current hypothesis states that hypoxic chemoreception is mediated by acid formation in the chemoreceptor cells (Hanson et al., 1981, Eyzaguirre et al, 1983, Fidone and Gonzalez, 1986). Using exogenously supplied lumiphors and video imaging methodology, we tested this hypothesis by monitoring changes in microvascular oxygen pressure and intracellular pH with those of chemosensory activity during oxygen deprivation. These studies were conducted using an in vitro preparation of the cat carotid body that was perfused and superfused with cell-free Tyrode solution (Iturriaga et al, 1990).

It is possible that glutamate and other amino acids enhance the responsiveness of carotid body preparations by acting as substrates for cellular energy production. Amino acids are potential metabolic substrates because they can be converted to tricarboxylic acid cycle intermediates involved in ATP generation (Altman and Dittmer, 1973). A high concentration of glutamate (42 mM) was used in the carotid body superfused in vitro (Baron and Eyzaguirre, 1977). Glutamate apparently helped to maintain responsiveness of the chemoreceptors. However, the extent to which glutamate was used as a metabolic substrate is unclear because the 5.5 mM glucose that was also present may have been an adequate substrate. Consistent with the hypothesis that glutamate and other amino acids are used as metabolic substrates by the carotid body is that vascularly isolated carotid body preparations perfused with saline solutions containing 5.5 mM glucose but lacking amino acids manifest progressive deterioration in chemosensory responsiveness (Joels and Neil, 1968; O’Regan, 1973).

A current hypothesis states that carotid body (CB) chemoreception is ultimately mediated by the release of neurotransmitters from the glomus cell1. One of the putative neurotransmitters is dopamine which is present in glomus cell dense-core vesicles1 and is released in response to hypoxia2, low pH and hypercapnia3. In the cat CB, however, exogenous dopamine inhibits chemosensory activity in vivo 4,5,6 and in vitro 7. Elucidation of the role of dopamine in CB chemoreception is further complicated by the finding that haloperidol, a D2 dopaminergic antagonist, increases the chemosensitivity of the CB to hypoxia4 and hypercapnia4,8 in vivo, but decreases the activity and the response to acetylcholine in vitro 9. Based on their in vitro observations, Nolan et al.9 proposed an excitatory role for dopamine. However, they did not use other stimuli to test if the effects of haloperidol are specific to O2 and CO2 chemoreception. In order to test this possibility we studied the effects of haloperidol on the chemosensory responses in vitro to hypoxia, CO2, dopamine, sodium cyanide and nicotine.

Measurements of oxygen pressures within the carotid body have been of particular importance for elucidating the mechanism(s) of oxygen chemoreception. An optical technique based on the quenching of phosphorescence by oxygen that is capable of imaging oxygen within the microvasculature has been developed (Rumsey et al, 1988, Wilson et al, 1991). These optical measurements are non-invasive and in particular have the advantage of determining oxygen pressures throughout the entire tissue. We have previously applied this technology to the study of the isolated perfused/superfused cat carotid body and oxygen chemoreception (Rumsey et al, 1991), relying primarily on imaging of phosphorescence intensity. Although the latter is quite useful for following dynamic changes in oxygen pressures, calculation of oxygen pressure is only qualitative. In addition, the carotid body was perfused and superfuised with cell-free media (Iturriaga et al, 1991). In the present report, we describe measurements of oxygen pressure determined from images in phosphorescence lifetimes (Wilson et al, 1991) in the cat carotid body using an in vivo preparation (Lahiri and DeLaney, 1975). These determinations were compared to those obtained previously with the in vitro preparation, thereby permitting comparison of the oxygenation of this organ in the presence and absence of blood, respectively. In both cases, chemosensory discharge was recorded in order to provide measurements of the expression of oxygen chemoreception.

Background Secretion of gonadotropin-releasing hormone (GnRH) produced in neurons in the basal forebrain is the primary regulator of reproductive maturation and function in mammals. Peptidergic signals relating to circadian timing and energy balance are an important influence on the reproductive axis. The aim of this study was to investigate the innervation of GnRH neurons by peptidergic neurons. Methodology/Principal Findings Immunohistochemistry and confocal microscopy were used to detect appositions of peptidergic fibers (NPY, β-endorphin, MCH) associated with energy balance and metabolic status in transgenic mice expressing a green fluorescent protein reporter construct in GnRH neurons. The frequency of these appositions was compared to those of vasoactive intestinal peptide (VIP), a hypothalamic neuropeptide likely to convey circadian timing information to the GnRH secretory system. The majority of GnRH neurons (73–87%) were closely apposed by fibers expressing NPY, β-endorphin, or MCH, and a significant proportion of GnRH neurons (28%) also had close contacts with VIP-ir fibers. Conclusions/Significance It is concluded that GnRH neurons in the mouse receive a high frequency of direct modulatory inputs from multiple hypothalamic peptide systems known to be important in conveying circadian information and signalling energy balance.

An in vitro perfused carotid body preparation was developed to study its chemosensory responses to physiological and pharmacological stimuli. The carotid bifurcation with the carotid body was vascularly isolated and excised from pentobarbital sodium-anesthetized cats. The CB was perfused in a chamber by gravity (80 Torr) with modified Tyrode's solution (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-NaOH at pH 7.40) equilibrated at a given Po2 and superfused with the same medium at (Po2 of 20 Torr). The temperature was maintained at 35.5 +/- 0.5 degrees C. The frequency of chemosensory discharges (CD) was recorded from the whole carotid sinus nerve (n = 24), and the responses were tested by repeated interruptions of perfusate flow (SF), perfusion with hypoxic medium, and injections of nicotine and cyanide (0.1 nmol to 1 mumol) and hypercapnic medium. During hyperoxic perfusion, SF resulted in a sigmoidal increase in CD, reaching a maximum that was 23.6 +/- 4.4-fold greater than the basal activity. Restoration of flow returned CD promptly to basal values. After normoxic perfusion, SF led to a similar maximal activity more rapidly, but the duration was shorter. Reduction of the perfusate PO2 (Po2 from 450 Torr to 150, 30, and less than 10 Torr) caused a nonlinear increase in CD. CO2 stimuli (PCo2 38-110 Torr) resulted in a linear increase in CD. Nicotine or cyanide increased CD in a dose-dependent manner. The preparation retained its initial responsiveness for 2-3 h, making extensive experimental studies feasible.(ABSTRACT TRUNCATED AT 250 WORDS)

Perfusion of individual organs of the body in the isolated state is a powerful tool for the study of their integrated function. This approach has provided, for example, a wealth of knowledge on the relationship between metabolism and function in several tissues, including heart, liver, kidney, and skeletal muscle. The carotid body, however, has proven unreliable as an in vitro perfused model.

In hirudinid leeches, the segmental ganglia associated with the sexual organs contain several hundred more neurons than other midbody ganglia. To determine whether this difference arises by differential cell addition or by differential cell death, cell counts were made in several segmental ganglia during the course of embryonic and postembryonic development. The results show that all ganglia behave equally in early development. In each case, at least 10-20% more cells than will make up the adult complement of about 400 neurons is generated, and by about 20 days of embryonic development cell loss brings the number down to about 400 cells. By about 30 days, when animals emerge from their cocoons, additional cells have begun to appear in the sex ganglia. The number of extra cells continue to increase gradually over the next several months, until the adult number of 600-700 neurons is attained. These observations indicate that at least some segmental differences in the size of neuronal populations are due to differential cell proliferation and that these differences can arise quite late in the maturation of an animal.