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The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2020 Li, Su, Zhao, Li, Xu, Gao, Sun, Yang, Jin and Ke.)

Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones., (© 2016 McMahon et al.)

The pituitary gland is an endocrine organ that is developmentally derived from a fold in the oral ectoderm and a juxtaposed fold in the neural ectoderm. Here, we show that the absence of Vax1, a homeodomain transcription factor known for its role in eye and optic chiasm development, causes the rostral oral ectoderm to form an ectopic fold that eventually develops into a separate second pituitary with all the pituitary cell types and neuronal fibers characteristic of the normal pituitary. The induction of the second pituitary is associated with a localized ectopic expression of Fgf10, a gene encoding a growth factor known to recruit oral ectodermal cells into the pituitary. Interestingly, there are rare cases of pituitary duplications in humans that are also associated with optic nerve dysplasia, suggesting that VAX1 might be involved in the pathogenesis of this disorder.

We elucidated the structure of the principle factors regulating the initiation of the acute stress response in common carp: corticotrophin-releasing hormone (CRH), CRH-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP). Phylogenetic analyses reveal that these proteins are evolutionarily well conserved in vertebrates. CRH and CRH-BP expression are not co-localised in the same hypothalamic perikarya. On the contrary, CRH-BP expression is limited to the perimeter of the nucleus preopticus (NPO), but is abundant in other regions, including an area directly rostral from, and in close proximity to, the NPO. Despite the lack of co-expression, the nerve fibres projecting onto both the rostral pars distalis (rPD) as well as the large fibre bundles projecting onto the pars intermedia (PI) contain CRH as well as CRH-BP, suggesting that both ACTH release from the rPD as well as the release of PI melanotrope content is regulated via CRH and CRH-BP. Finally, we show via real-time quantitative PCR that expression of hypothalamic CRH and CRH-BP following a 24 h restraint significantly increases, whereas PD CRH-R1 expression decreases; this reflects desensitisation of the PD for hypothalamic CRH output. We conclude that these factors are actively involved in the regulation of acute stress responses in the teleost fish.

The release of a bioassayable form of growth hormone (BGH), distinct from growth hormone as measured by immunoassay (IGH), from the rat pituitary into the blood is differentially regulated by afferent input from fast and slow skeletal muscles. Specifically, activation of low-threshold fast muscle afferents for 15 min increased plasma BGH by 217 and 295% and decreased pituitary BGH by 68 and 45% in male and female rats, respectively. In contrast, activation of slow muscle afferents inhibited BGH release, decreasing plasma BGH by approximately 60% and increasing pituitary BGH by 30-50% in male rats. Female rats from which food had been withheld for approximately 12 h had elevated basal plasma BGH levels, which then were decreased by 81% after slow muscle nerve stimulation. Plasma IGH concentrations were unchanged after any nerve stimulation condition. These results demonstrate that regulation of BGH release can be differentially mediated through low-threshold afferent inputs from fast or slow skeletal muscle. Furthermore, the results indicate that BGH responses are independent of gender or feeding status.

1. A novel slowly activating voltage-dependent K+ current was observed in isolated nerve terminals from rat neurohypophysis using the whole-cell configuration of the patch-clamp technique. 2. The activation kinetics of the slow current could be fitted assuming Hodgkin--Huxley-type kinetics, an exponential, n, of 1.3 and activation time constants decreasing from 4 s at -50 mV to 0.7s at +40 mV. 3. A positive shift of reversal potential was observed when [K+] was increased in the bath solution. The current is carried mainly but not exclusively by K+ ions. 4. When intracellular free [Mg2+] was low (approximately 60 microM), average current density was 74 pA pF-1 at membrane potentials around 0 mV. In 83% of nerve terminals current amplitude was > 10 pA pF-1. 5. The slow current was never observed when the pipette contained 4.6 mM free Mg2+. At a physiological level of free Mg2+ (0.5 mM) the average current density was 16 pA pF-1. 6. When nerve terminals were analysed after patch-clamp experiments for vasopressin content by immunodetection, no difference in current amplitude was found between the terminals containing vasopressin and all analysed terminals. 7. The voltage dependence of activation was fitted by a Boltzmann equation giving a half-activation potential of -37 mV and a slope factor of about 9 mV. 8. Tail current deactivation kinetics was biexponential with time constants of 0.12 and 1.5s. Kinetics was dependent on the duration of the activating pulse. 9. Noise analysis of the slow current indicated a single-channel current of 0.33 pA at +6 mV, corresponding to a single-channel conductance of 4.3 pS. 10. This is the first demonstration of a current similar to the slow K+ current, IKs, in a neurone, suggesting that a protein similar to the IKs-inducing channel protein IsK (minK) may be present in peptidergic nerve terminals. 11. The activation properties are consistent with a role of the slow current in inhibition of excitability, at least at the level of the nerve terminal.

The retinoblastoma gene (RB) is the prototypic tumor suppressor. Studies to date have demonstrated cancer suppression with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb +/- mice. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb +/- mice were treated with a recombinant adenovirus carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, reestablished innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life spans of treated animals.

To better understand the cell lineage-specific character of retinoblastoma (Rb) gene inactivation during tumor formation, the earliest stages of spontaneous melanotroph carcinogenesis in Rb+/- heterozygous mice have been subjected to sequential analyses. The first atypical cells are detected in the pituitary intermediate lobe during a period corresponding to the cessation of melanotroph proliferation between 35 and 60 days after birth. Atypical cells contain no wild-type copy of the Rb gene and synchronously form early atypical proliferates (EAP) in the subsequent 30-60 day period. In contrast to surrounding mature melanotrophs with the wild-type Rb gene, Rb-negative cells in EAP continue to proliferate well past postnatal day 60, and fail to be innervated by growth inhibitory dopaminergic nerve terminals. Atypical melanotrophs remain competent for dopamine D2 receptor stimulation and undergo S-phase apoptosis in close proximity to nerve terminals. These results indicate a key role for the Rb protein in the onset of neuron-neuroendocrine cell interactions. This role may explain cell-type-specific neuroendocrine carcinogenesis associated with inactivation of the ubiquitously expressed Rb gene.

Recent immunohistochemical evidence from the rat, indicating that gamma-aminobutyric acid (GABA)-containing fibres of central nervous origin project to the pars intermedia of the pituitary1,2, prompts inquiry into the function of this innervation. There is electrophysiological evidence that GABA acts directly on melanotrophs isolated from rat, through bicuculline-blockable receptors, to increase Cl- conductance and thereby drive the membrane potential towards the Cl- equilibrium potential in these cells, resulting in depolarization at rest or reduction of the depolarization caused by excess K+ (ref. 3). As voltage-dependent Ca channels can participate in the regulation of secretion in these cells4, we have now examined the effect of GABA on hormone output and find that it first stimulates and then inhibits spontaneous secretion of melanocyte-stimulating hormone (MSH) and inhibits K+-evoked secretion. Moreover, our pharmacological evidence suggests that similar receptors are involved in the secretory and the electrophysiological responses. A function of the GABAergic innervation may therefore be to regulate hormone output by acting directly on the melanotrophs, and this regulation may be affected by the changes in electrical properties induced by GABA.

Immunoperoxidase histochemical investigations on pig hypophysis show that CRF immunoreactive nerve terminals are distributed around the blood vessels in the posterior lobe, particularly in its proximal part adjacent to the intermediate lobe. CRF immunoreactive nerve fibers are also seen in the intermediate lobe. The distribution of the CRF-containing nerve in the posterior lobe was found to be similar to that of the vasopressin-containing nerve terminals. The functional significance of the presence of CRF-containing nerve fibers representing a new component of the hypothalamo-hypophyseal neurosecretory system is discussed.

Activity of glutamic acid decarboxylase GluDCase, the biosynthetic enzyme of gamma-aminobutyric acid (GABA) was measured in low-speed homogenate supernatant of the neural and intermediate (neurointermediate) lobe (28--30 pmol of CO2 per microgram of protein per hr) and of the anterior lobe (2--4 pmol of CO2 per microgram of protein per hr). In the neurointermediate lobe, stalk transection reduced the GluDCase activity by more than 95%. By using an antiserum to rat brain GluDCase and the unlabeled antibody--peroxidase method of Sternberger, GluDCase immunoreactivity was localized in many terminals within the neurointermediate lobe of the hypophysis. In pars intermedia, immunoreactive terminals occurred in apposition to secretory cells and to glial cells and were near nonimmunoreactive axonal profiles; in pars neuralis they were apposed to pituicytes and to unlabeled axons including the neurosecretory terminals and were along fenestrated portal capillaries. GluDCase immunoreactive axons terminals exhibited diverse morphological features and would not have been identified as a distinct population without the GluDCase antiserum. No GluDCase-immunoreactivity was found in the anterior pituitary lobe. Stalk transection abolished GluDCase immunoreactivity in the neurointermediate lobe. These data provide biochemical and morphological evidence for a central GABAergic innervation of neural and intermediate lobes of the hypophysis.

Short-term in vitro incubation of hypothalamo-hypophyseal tissue from young rats was undertaken to discern more clearly the functional relationship between putative dopaminergic neural projections in the pars intermedia and the secretory activity of melanophore stimulating hormone (MSH). This explant consisted of a portion of the mediobasal hypothalamus containing the dopamine neurone cell bodies of interest, with the attached pituitary neuro-intermediate lobe (n.i.l.). The n.i.l. was inserted into the end of a 1 mm diameter tube attached to a perfusion pump which allowed uninterrupted sampling of medium neighbouring the n.i.l. A 'real-time' analysis of hormone secretion was obtained by immediately and continuously bioassaying for MSH. A bipolar stimulating electrode was placed on the ventral floor of the mediobasal hypothalamus either directly on the arcuate nucleus, median eminence or infundibular stalk. Electrical stimulation for 5 min (0.1-20.0 Hz) caused a transient inhibition of basal MSH secretion, while continuous stimulation (0.1-5.0 Hz) led to a much greater, long-term, reversible inhibition. In the latter, the degree of inhibition was generally dependent on stimulation rate up to a maximum at 5 Hz. Application of the dopamine D2 receptor antagonist, 1-sulpiride (0.001-0.1 microM) to the perfusion medium not only completely and reversibly blocked the stimulus-induced inhibition of MSH release but by itself, significantly increased the basal secretion rate. Applied to the isolated n.i.l., 1-sulpiride did not alter release but did prevent the inhibitory response caused by exogenously applied dopamine (0.1 microM). The gamma-aminobutyric acid receptor antagonist, bicuculline (0.01-1.0 microM), had no effect on any of the parameters studied. In explants, cutting the infundibular stalk linking the mediobasal hypothalamus with the n.i.l., mimicked the effects of 1-sulpiride by interrupting impulse flow to the gland. Thus, electrical stimulation of hypothalamic neurones in these explants apparently causes a release of dopamine from nerve terminals in the pars intermedia to inhibit MSH secretion and perhaps other pro-opiomelanocortin-derived peptides as well.

Control of the secretion of ACTH appears to involve several mechanisms. It is dependent upon the release, from neurones in the hypothalamus, of corticotrophin releasing factor, which is probably a complex including CRF-41 and vasopressin. The secretion of the complex is influenced by various central inhibitory and excitatory nervous pathways. The releasing factor is conveyed by the hypophyseal portal vessels to the adenohypophysis where it stimulates the secretion of ACTH. The corticosteroids exert a profound control over the basal secretion of ACTH, but only modulate the pituitary adrenocorticotrophic response to stress. The mechanisms which may be involved are summarized in figure 8.