Neurosecretory Pathway Research Articles

Cell-specific expression and individual function of prohormone convertase PC1/3 in Tribolium larval growth highlights major evolutionary changes between beetle and fly neuroendocrine systems

BackgroundThe insect neuroendocrine system acts in the regulation of physiology, development and growth. Molecular evolution of this system hence has the potential to allow for major biological differences between insect groups. Two prohormone convertases, PC1/3 and PC2, are found in animals and both function in the processing of neuropeptide precursors in the vertebrate neurosecretory pathway. Whereas PC2-function is conserved between the fly Drosophila and vertebrates, ancestral PC1/3 was lost in the fly lineage and has not been functionally studied in any protostome.ResultsIn order to understand its original functions and the changes accompanying the gene loss in the fly, we investigated PC1/3 and PC2 expression and function in the beetle Tribolium castaneum. We found that PC2 is broadly expressed in the nervous system, whereas surprisingly, PC1/3 expression is restricted to specific cell groups in the posterior brain and suboesophageal ganglion. Both proteases have parallel but non-redundant functions in adult beetles’ viability and fertility. Female infertility following RNAi is caused by a failure to deposit sufficient yolk to the developing oocytes. Larval RNAi against PC2 produced moulting defects where the larvae were not able to shed their old cuticle. This ecdysis phenotype was also observed in a small subset of PC1/3 knockdown larvae and was strongest in a double knockdown. Unexpectedly, most PC1/3-RNAi larvae showed strongly reduced growth, but went through larval moults despite minimal to zero weight gain.ConclusionsThe cell type-specific expression of PC1/3 and its essential requirement for larval growth highlight the important role of this gene within the insect neuroendocrine system. Genomic conservation in most insect groups suggests that it has a comparable individual function in other insects as well, which has been replaced by alternative mechanisms in flies.

Chromogranin A in the early steps of the neurosecretory pathway.

Chromogranin A (CgA) is a soluble glycoprotein stored with hormones and neuropeptides in secretory granules (SG) of most (neuro)endocrine cells and neurons. Since its discovery in 1967, many studies have reported its structural characteristics, biological roles, and mechanisms of action. Indeed, CgA is both a precursor of various biologically active peptides and a granulogenic protein regulating the storage and secretion of hormones and neuropeptides. This review emphasizes the findings and theoretical concepts around the CgA-linked molecular machinery controlling hormone/neuropeptide aggregation and the interaction of CgA-hormone/neuropeptide aggregates with the trans-Golgi membrane to allow hormone/neuropeptide targeting and SG biogenesis. We will also discuss the intriguing alteration of CgA expression and secretion in various neurological disorders, which could provide insights to elucidate the molecular mechanisms underlying these pathophysiological conditions.

HID-1, a New Component of the Peptidergic Signaling Pathway

hid-1 was originally identified as a Caenorhabditis elegans gene encoding a novel conserved protein that regulates the decision to enter into the enduring dauer larval stage. We isolated a novel allele of hid-1 in a forward genetic screen for mutants mislocalizing RBF-1 rabphilin, a RAB-27 effector. Here we demonstrate that HID-1 functions in the nervous system to regulate neuromuscular signaling and in the intestine to regulate the defecation motor program. We further show that a conserved N-terminal myristoylated motif of both invertebrate and vertebrate HID-1 is essential for its association with intracellular membranes in nematodes and PC12 cells. C. elegans neuronal HID-1 resides on intracellular membranes in neuronal cell somas; however, the kinesin UNC-104 also transports HID-1 to synaptic regions. HID-1 accumulates in the axons of unc-13 and unc-31 mutants, suggesting it is associated with neurosecretory vesicles. Consistent with this, genetic studies place HID-1 in a peptidergic signaling pathway. Finally, a hid-1 null mutation reduces the levels of endogenous neuropeptides and alters the secretion of fluorescent-tagged cargos derived from neuronal and intestinal dense core vesicles (DCVs). Taken together, our findings indicate that HID-1 is a novel component of a DCV-based neurosecretory pathway and that it regulates one or more aspects of the biogenesis, maturation, or trafficking of DCVs.

Regulation by Rab3A of an endogenous modulator of neurotransmitter release at mouse motor nerve endings.

Rab3A, a small GTP-binding protein attached to synaptic vesicles, has been implicated in several stages in the process of neurosecretion, including a late stage occurring just prior to the actual release of neurotransmitter. The inhibitory neuromodulator adenosine also targets a late step in the neurosecretory pathway. We thus compared neuromuscular junctions from adult Rab3A(-/-) mutant mice with those from wild-type mice with respect to: (a) the basic electrophysiological correlates of neurotransmitter release at different stimulation frequencies, and (b) the actions of exogenous and endogenous adenosine on neurotransmitter release in normal calcium solutions. Neither the spontaneous quantal release of acetylcholine (ACh) nor basal evoked ACh release (0.05 Hz) differed between the mutant and wild-type mice. At 50-100 Hz stimulation (10-19 stimuli), facilitation of release was observed in the mutant mice but not in wild-type, followed by a depression of ACh release in both strains. ACh release at the end of the stimulus train in the mutant mouse was approximately double that of the wild-type mouse. The threshold concentration for inhibition of ACh release by exogenous adenosine was over 20-fold lower in the mutant mouse than in the wild-type mouse. The adenosine A(1) receptor antagonist 8-cyclopentyltheophylline (CPT) increased ACh release (0.05-1 Hz stimulation) in the mutant mouse under conditions in which it had no effect in the wild-type mouse. CPT had no effect on the pattern of responses recorded during repetitive stimulation in either strain. The results suggest that Rab3A reduces the potency of adenosine as an endogenous mediator of neuromuscular depression.

Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor.

A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. We show here that two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation.

Immunohistochemical demonstration of oxytocin-like, neuropeptide Y-like and gonadotropin-releasing hormone-like substances in the hypothalamo-hypophysial system of ayu,plecoglossus altivelis altivelis, in osmotically different environments

To ascertain the role of neuropeptides on the hypothalamo-hypophysial system of a fish in osmotically different environments, an immunohistochemical study of oxytocin (OXT), neuropeptide Y (NPY) and gonadotropin-releasing hormone (GnRH) was carried out on the anadromous salmonoid fish,Plecoglossus altivelis altivelis, commonly known as Ayu. River fish caught were acclimatized in a freshwater aquarium, half of them being subsequently kept as a control group and the remainder being transferred to a sea water aquarium, through 1/3 diluted sea water, as an experimental group. OXT-like immunoreactivity as demonstrated in the neurosecretory pathway, having the same pattern was that shown by aldehyde fuchsin staining. Noticeably, a mass of nucleus preopticus (NPO) and a marginal portion of the pars nervosa in the control group became strongly immunoreactive, whereas a very weak reaction was obtained in the sea water-retained fish, suggesting the release of the labelled substance. In the latter, NPY-like substance was widely distributed in the brain without NPO, with the positive substance being dense in the terminal rami of the pars nervosa bordering the pars distalis. However, no remarkable difference in GnRH-like and NPY-like immunoreactivities in the hypothalamo-hypophysial system was apparent between the two groups. These results suggested that OXT (probably isotocin)-like substance may play a role in osmoregulation.

Localization of a Crustacean Hyperglycemic Hormone-like Immunoreactivity in the Neuroendocrine System ofEuscorpius carpathicus(L.) (Scorpionida, Chactidae)

The neuroendocrine system ofEuscorpius carpathicuswas immunohistochemically localized using a polyclonal antiserum raised against a purifiedHomarus americanuscrustacean hyperglycemic hormone (Hoa-cHHA). There were cross-reactions inE. carpathicusprocerebral and subesophageal neurosecretory cells, neurohemal organs, and intra- and extraganglionic neurosecretory tracts. Among the neurohemal structures, the Kwartirnikov's organ, the Tropfenkomplex, and the coxal disc reacted strongly. InEuscorpius,the differing results between adults and juveniles suggest neurosecretory variations related to developmental stage. These immunohistochemical observations suggest the presence of substances related to the cHH in scorpions; however, in this heterologous system, it is not at present possible to assess physiological significance.

A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans.

A pheromone-induced neurosecretory pathway in Caenorhabditis elegans triggers developmental arrest and an increase in longevity at the dauer diapause stage. The gene age-1 is required for non-dauer development and normal senescence. age-1 encodes a homologue of mammalian phosphatidylinositol-3-OH kinase (PI(3)K) catalytic subunits. Lack of both maternal and zygotic age-1 activity causes dauer formation, whereas animals with maternal but not zygotic age-1 activity develop as non-dauers that live more than twice as long as normal. These data suggest that phosphatidylinositol signalling mediated by AGE-1 protein controls lifespan and the dauer diapause decision.

Neuronal pathways of classical crustacean neurohormones in the central nervous system of the woodlouse, Oniscus asellus (L.)

Neuropeptide-immunoreactive neurons have been mapped by immunocytochemistry in whole-mount preparations and sections of the central nervous system of Oniscus asellus . We tested rabbit antisera against decapod crustacean hyperglycemic hormone (CHH), moult inhibiting hormone (MIH ), pigment dispersing hormone (PDH) and red pigment concentrating hormone (RPCH). four CHH- and three PDH-immunoreactive neurons localized in the superior median protocerebrum of the brain constitute neurosecretory pathways to the neurohaemal sinus gland. No immunoreactive structures have been detected with an antiserum against MIH of Carcinus maenus . Another, newly identified neurosecretory pathway is formed by a group of RPCH-immunoreactive neurons in the mandibular ganglion. These neurons project to the neurohaemal lateral cephalic nerve plexus, further PDH- and RPCH-immunoreactive neurons and fibres occur in the brain and the ventral nerve cord (VNC). Two groups of PDH-immunoreactive neurons supply brain and optic lobe neuropils, the bases of the ommatidia, and probably give rise to descending fibres innervating all VNC-neuropils. Two groups and five individuals of RPCH-immunoreactive neurons that innervate several brain neuropils or occur as ascending neurons in the VNC have been reconstructed. The CHH-immunoreactive neurons, and distinct types of PDH- and RPCH-immunoreactive neurons obviously belong to classical hormone-producing neurosecretory pathways. At least the CHH-immunoreactive cells seem to be part of an isopod homologue of the decapod X-organ. The existence of other PDH- and RPCH-immunoreactive interneurons suggests additional functions of these peptides as neurotransmitters or neuromodulators, which is in agreement with similar observations in the decapod central nervous system.

Inhibition of high voltage-activated Ca2+ currents from cultured sensory neurones by a novel insect peptide.

PMP-D2, a novel 35 amino acid peptide isolated from the brain of the locust Locusta migratoria, is localised specifically in neurosecretory cells and nerve tracts of the Pars intercerebralis. When PMP-D2 is applied onto rat sensory neurones it blocks high voltage-activated inward Ca2+ currents at concentrations ranging from 0.1 mu M to 10 mu M. The inhibitory effect of PMP-D2 is more marked on the sustained inward Ca2+ current measured at the end of 100 ms voltage step commands than on the maximum inward Ca2+ current. These results suggests that PMP-D2 may differentially inhibit the two components of the high voltage-activated inward Ca2+ currents of rat sensory neurones.

Aggregation of vasopressin mRNA in a subset of axonal swellings of the median eminence and posterior pituitary: light and electron microscopic evidence

The mRNA encoding vasopressin has recently been documented within the magnocellular hypothalamo-neurohypophyseal projections of the rat such as the median eminence (ME) and the posterior pituitary (PP), suggesting the possibility of its axonal transport. To address the origin of this mRNA and to investigate the functional significance of this unexpected axonal transport of mRNA, we have examined its subcellular localization within both magnocellular perikarya and their axonal projections. For this purpose, we have used nonradioactive in situ hybridization techniques in order to localize the vasopressin mRNA with precision at the ultrastructural level in magnocellular perikarya, dendrites, and axons from control, salt-loaded, and lactating rats. This approach permitted us to demonstrate directly the axonal localization of vasopressin mRNA. Moreover, we were able to obtain novel information concerning vasopressin mRNA compartmentation within both perikarya and axons. At both light and electron microscopic levels, we observed vasopressin mRNA-containing cells in the hypothalamic magnocellular cell body groups, but not in the ME or in the PP. When vasopressin mRNA was detected in medium-size dendrites, it was always associated with the rough endoplasmic reticulum (RER). Within the labeled magnocellular perikarya, the abundant vasopressin mRNA was mainly associated with discrete areas of the RER. However, vasopressin mRNA was never detected in the Golgi apparatus or in association with neurosecretory granules, in perikarya or axons. These data suggest that vasopressin mRNA translation is restricted to certain segments within the RER, and that axonal transport of vasopressin mRNA does not involve the classical neurosecretory pathway, via the Golgi apparatus and the neurosecretory granules, as has been proposed. Within the magnocellular neuron axons, vasopressin mRNA could be detected only in a subset of axonal swellings, all of which were confined to the internal layer of the ME and the PP. The mRNA-containing swellings were numerous in 7 d salt-loaded animals, less abundant in lactating animals, and almost undetectable in control animals. In all groups of animals, no vasopressin mRNA was detectable in any other region of the magnocellular neuron axons, including undilated axonal segments or varicose swellings. These results strongly suggest that, under physiological activation such as chronic salt loading, axonal vasopressin mRNA is increased and becomes aggregated in a selected subset of swellings of the ME and the PP. Furthermore, these data indicate that along the magnocellular neuron axons, the swellings may differ in their biochemical and functional features. Further analysis focused on the mRNA-accumulating swellings may illuminate the function of RNA within the axonal compartment.

Characterization of the calcium and chloride [ 3H]glutamate binding site in crude synaptic membranes from human brain tissue

1. The specific binding properties of [ 3H]glutamate to crude synaptic membranes (CSM) from postmortem human brain were studied. 2. Equilibrium binding analysis of [ 3H]glutamate binding to CSM from human brain cortex revealed a K D = 110 ± 12 nM and a B max = 27 ± 4 pmol/mg protein). 3. Calcium increased the number of binding sites, B max = 44 ± 6 pmol/mg protein, without a significant change in the affinity constant, K D = 95 ± 10 nM. 4. The dissociation constant of the [ 3H]glutamate bound to human CSM was 4.0 ± 0.4 min −1 ( n = 3). 5. The relative potencies of glutamate analogs and 2-amino-4-phosphonobutyric acid (APB) to compete for the glutamate binding sites, in human CSM, were glutamate > quisqualate = ibotenic acid > APB ⪢ α-amino-3-hydroxy-5-methyl-4-isoxozolepropionate acid. 6. The glutamate specific binding in CSM from postmortem human brain was particularly rich in the gyrus hippocampus, nucleus accumbens, thalamus and frontal cortex. 7. This glutamate binding protein is related, probably, to a presynaptic neurosecretory pathway.

Distribution and morphometric characteristics of oxytocin- and vasopressin-immunoreactive neurons in the rabbit hypothalamus.

The distribution, morphological features, and morphometric characteristics of cell bodies producing oxytocin (OT) and vasopressin (AVP) were studied in the rabbit hypothalamus by means of a conventional immunoperoxidase method. The aim of the present study was to determine the existence or not of a species-specific OT-cell group that might be involved in the dense OT innervation of the intermediate lobe in the leporidae. No OT-cell group clearly distinct from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei was found, even in colchicine-treated animals. Most immunoreactive perikarya were found within these nuclei. In addition, small AVP neurons occurred in the suprachiasmatic nucleus. In the SON, the predominant, tightly packed AVP cells occupied the ventral part of the nucleus, whereas OT neurons were dorsolaterally located. The PVN presented a loose organization without any obvious subdivision. OT cells, which predominated, occupied the medial part of the nucleus. The PVN had a prominent rostral anterobasal extension composed mainly of OT cells. Laterally to the nucleus, numerous large AVP neurons, with few and smaller OT cells, dispersed along the neurosecretory tract without forming definite cell clusters. AVP cell bodies had a rough granular aspect contrasting with the smooth and fine one of OT cells. Spinelike processes were rarely observed on the perikarya, except on large scattered AVP neurons, but frequently covered the proximal dendrites of both types of neurons. Throughout the hypothalamus, OT neurons had definitely smaller mean somal areas and were more homogeneous in size than AVP cells.

Commissural ring nerve: A female-specific neurosecretory tract supplied by bifurcating median neurons in the cockroach Periplaneta americana (L.) and the cricket Teleogryllus commodus (Walker)

In the American cockroach, Periplaneta americana, and the Australian field cricket, Teleogryllus commodus, the two nerves supplying the bases of the cerci are joined by a branch that crosses behind the last abdominal ganglion. This commissural ring nerve is restricted to females, and it contains many axons filled with granular and agranular vesicles. The axons stem from somata located within the ganglion. There are one (Periplaneta) or two (Teleogryllus) groups of median neurons with bilaterally symmetrical bifurcations, and a group of postero-ventral neurons on each side. In T. commodus, these neurons are distinct from others associated with the cerci. In the two species, the ring nerve neurons contribute to a neuropile near the root of each cereal nerve. The bifurcating median neurons arborize on both sides before entering the ring nerve, while the postero-ventral ones branch more extensively ipsilateral to their somata. The possibilities are discussed that the bifurcating neurons may be homologous to dorsal unpaired median neurons, and that the ring nerve may be a neurohemal area.

Course of neurosecretory tracts within hypothalamus in a snow trout,Schizothorax niger (Heckel) byin situ technique

The origin and course of hypothalamic neurosecretory tracts is demonstrated byin situ staining procedure. Both Pars parvocellularis and Pars magnocellularis, components of nucleus preopticus, contribute to the formation of the right and left main neurosecretory tract. Anteriorly the tract is loosely assembled and formed of 3 subsidiary tracts which later join at the level of horizontal commissure to form a thick consolidated single tract. From the main tract several bundles of axons are given towards the mid-ventral region at the point of curvature to form ventromedian tracts. The ventromedial and the main tract of each side jointly enter the pituitary, anterio-dorsally.

Identification of the Bursicon-Containing Neurones in Abdominal Ganglia of the Tobacco Hornworm, Manduca Sexta

ABSTRACT The abdominal ganglion neurones responsible for the secretion of the peptide hormone bursicon from the transverse nerves of the tobacco hornworm Manduca sexta have been localized. Each unfused abdominal ganglion produced bursicon during adult development. The hormone was then transported to the next posterior transverse nerve for subsequent release following adult emergence. Of the two efferent pathways that connect ganglia to posterior transverse nerves, only one had ultrastructural features consistent with a neurosecretory function. All axons in this pathway contained numerous, elementary, dense-cored granules with diameters ranging from 100 to 300 nm. In pharate adults, cobalt back-filling of this neurosecretory pathway revealed two groups of cell bodies in abdominal ganglia : three dorso-laterally and eight at the mid-line. Hormonal activity was present in lateral regions of the ganglion but not in mid-line regions. In the dorso-lateral region, four blueish cell bodies could be seen in the living tissue. The two combined clusters of four cells from both sides of the ganglion contained more than 60% of the bursicon activity present in the entire ganglion. More careful dissection of the cluster detected the presence of activity in cell pairs and individual members. Individual dye-fills of these blue cells demonstrated that three of these were the three lateral neurones that were back-filled from the transverse nerve. The fourth blue cell may be identical to a neurone whose axon travels in the descending, contralateral connective and exits the nervous system via the next posterior ganglion. On the basis of dendritic geometry, each of the neurones in the bursicon cell cluster could be identified as a unique, individual neurone.

Skin-colour changes i the lizard, Anolis carolinensis, in response to localized electrical stimulation and lesions in the diencephalon.

A study was made of changes in skin colour in the lizard, Anolis carolinensis, in response to deep electrical stimulation at 0.2 mm intervals throughout the periventricular region of the diencephalon and the anterior brain stem. Double-barrelled glass microelectrodes with tip diameters of 3 microns were used. A 20 microA pulse-train consisting of a 500 Hz signal lasting for 1 s yielded localized responses. Skin darkening occurred only in response to stimulation delivered in the anterior and dorsal region of the diencephalon and skin lightening only in response to stimulation in a small area in the posterior and ventral region of the hypothalamus. Electrical lesions in the latter region resulted in permanent skin darkening. Surgical interruption of the hypothalamo-hypophysial neurosecretory tract did not block skin-colour change in response to dark or light backgrounds. It was concluded that MSH release is under tonic inhibitory control by hypothalamic neurones in Anolis. Both inhibitory and stimulatory neurones can be localized stereotaxically in the diencephalon and neither type corresponds with the neurosecretory neurones of the hypothalamo-hypophysial tract. The functional relationship between the stimulatory neurones and the inhibitory neurones and pars intermedia remains unclear.

Secretion of axonally transported neural peptides from the nervous system of Aplysia.

The possibility that proteins reaching the abdominal ganglion of Aplysia by axonal transport from the circumesophageal ganglia might be subject to secretion in that structure was examined. Transported labeled protein was found to be released from the abdominal ganglion; such release was enhanced by exposure to a high K+ medium and by electrical stimulation of the transporting axons. Stimulation of release was inhibited by lowering the Ca2+/Mg2+ ratio of the medium. The released material is predominantly of 1--2000 daltons in molecular weight and appears to have been derived from a group of transported peptides of about the same size. The possibility is raised that these data may reflect the existence of a peptidergic second-order neurosecretory pathway in this nervous system.

The neurosecretory hypothalamo-hindbrain pathway and its possible significance for the regulation of blood pressure and the milk-ejection reflex

1. In lactating rats and in rats deprived of water, the amount of neurosecretory material in the fibres of the neurosecretory hypothalamohindbrain pathway exceeds that in untreated control animals. Under these experimental conditions the pathway and its target regions can be well analysed by means of fluorescence and electron microscopic methods. 2. The axons belonging to the hypothalamo-hindbrain pathway originate from perikarya located in the caudal portion of the nucleus paraventricularis and also from a small group of perikarya in the caudo-lateral hypothalamus. On the way to the hindbrain the neurosecretory fibres join other fibre bundles of the mid- and hindbrain. 3. In the hindbrain most of the neurosecretory fibres terminate in the area of the nucleus tractus solitarii and in the area of the dorsal column nuclei. The axon terminals form synapses with other neurones. 4. Using cytochemical methods at the ultrastructural level (Naumann and Sterba, 1976), the authors were able to prove that the vesicles in the exohypothalamic fibres and in their synaptic terminals contain the same sort of material as the neurophysin vesicles in the posterior lobe of the hypophysis. 5. The most distinct increase in neurophysin was observed in lactating females which were separated from their sucklings after a normal lactation period of 15 days and killed four days thereafter, and in rats deprived of water for different time periods. 6. The relationship of the neurosecretory hypothalamo-hindbrain pathway to the nucleus tractus solitarii and to the dorsal column nuclei suggests that, functionally, there may be a correlation between the system of blood-pressure control and the milk ejection reflex.

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole, Alytes obstetricans Laur.

Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.