Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

<p>Abstract</p> <p>Background</p> <p>Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.</p> <p>Results</p> <p>To develop a new tool for the identification of <it>in vitro </it>enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (<it>penk1</it>) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the <it>penk1 </it>promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.</p> <p>Conclusions</p> <p>These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The <it>penk1</it>-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.</p

Influence of aging on the neurochemical organization of the rat paraventricular nucleus.

The paraventricular nucleus (PVH) of the hypothalamus is a key region for the integration of the autonomic and neuroendocrine mechanisms. This integration becomes less reliable with age. Some critical functions, such as eating and drinking, body-temperature regulation, autonomic and endocrine responses which regulate the cardiovascular system seem to be particularly affected by the aging-related processes. In this paper, we analysed by means of immunocytochemistry the neurochemical organization of the magnocellular and parvocellular component of the PVH in old male rats. The main results concerning the neurohormones and the carrier proteins are the following: a significant decrease in the number of the oxytocin- (OXY) like immunoreactive neurons of the medial and lateral parvocellular nuclei; a decrease in the vasopressin- (VAS) like immunoreactive neurons of the medial and lateral parvocellular nuclei and also of the medial magnocellular nucleus; a decrease in the neurophysin- (NRP) like immunoreactive neurons of the lateral parvocellular nucleus. We also found a decrease in the mean area of magnocellular OXY- and VAS-like immunoreactive neurons, a decrease in the extension of the dendritic tree sampled in the medial part of the nucleus; a decrease in the number of varicosities along the neurons coming from the PVH, and in the density of axons in the median eminence and in the vagal complex. The NRP-like immunoreactive structures in the substantia gelatinosa of the spinal cord of old rats were also decreased in respect to younger adult animals. Among the neuropeptides investigated (corticotropin-releasing factor, leu-enkephalin, somatostatin, cholecystokinin and neurotensin) we found a decrease in the leu-enkephalin-like immunoreactive neurons of the dorsal and medial parvocellular nuclei. Our data support--from a morphological point of view--the existence of involution processes in the neurochemical organization of the PVH during aging

Inhibin and activin in human fetal membranes: evidence for a local effect on prostaglandin release.

Trophoblast and fetal membranes (amnion and chorion) are the tissues forming the anatomical/functional interface between the mother and the fetus. The aim of the present study was to evaluate the presence and a possible effect of inhibin and activin in human amnion and chorion. The expression of inhibin/activin alpha, beta A, and beta B subunit messenger RNA (mRNA) and the localization of immunoreactive material were evaluated in amnion and chorion collected at term pregnancy by a Northern blot analysis and by immunohistochemistry. Amnion cells expressed the mRNA of the three inhibin/activin subunits, with the beta B message the most abundantly expressed. The epithelial layer of the amnion showed an intense fluorescent staining of beta B inhibin/activin subunit, and positive signals were also observed for the alpha and beta A subunits. Inhibin/activin alpha and beta A subunit mRNAs were found highly expressed in chorion. The cytotrophoblast of the chorion showed a positive staining for the three inhibin/activin subunit antisera. The addition of activin A increased the release of prostaglandin E2 from human amnion-derived cultured cells. The effect was dose and time dependent. Inhibin A did not induce significant changes of prostaglandin E2 release from amnion cells. The present results show that amnion and chorion produce inhibin and activin subunits and that activin stimulates the release of prostaglandin E2 from cultured amnion cells, suggesting a possible role of inhibin and activin in fetal membranes

Increase of neuropeptide Y-like immunoreactivity in the paraventricular nucleus of fasting rats.

The paraventricular nucleus (PVH) of the hypothalamus is a key region for the control of food intake. It presents a very high neuropeptide Y (NPY)-like positive innervation. In this paper we have studied the modifications of NPY-positive innervation in the PVH of 72 h starved rats vs control rats by means of semiquantitative immunocytochemistry. We observed a significant increase of NPY-like immunoreactivity in fasting rats. This result suggests a physiological role of NPY in the food intake regulation at the PVH leve

Inhibin and activin in human fetal membranes: evidence for a local effect on prostaglandin release.

Trophoblast and fetal membranes (amnion and chorion) are the tissues forming the anatomical/functional interface between the mother and the fetus. The aim of the present study was to evaluate the presence and a possible effect of inhibin and activin in human amnion and chorion. The expression of inhibin/activin alpha, beta A, and beta B subunit messenger RNA (mRNA) and the localization of immunoreactive material were evaluated in amnion and chorion collected at term pregnancy by a Northern blot analysis and by immunohistochemistry. Amnion cells expressed the mRNA of the three inhibin/activin subunits, with the beta B message the most abundantly expressed. The epithelial layer of the amnion showed an intense fluorescent staining of beta B inhibin/activin subunit, and positive signals were also observed for the alpha and beta A subunits. Inhibin/activin alpha and beta A subunit mRNAs were found highly expressed in chorion. The cytotrophoblast of the chorion showed a positive staining for the three inhibin/activin subunit antisera. The addition of activin A increased the release of prostaglandin E2 from human amnion-derived cultured cells. The effect was dose and time dependent. Inhibin A did not induce significant changes of prostaglandin E2 release from amnion cells. The present results show that amnion and chorion produce inhibin and activin subunits and that activin stimulates the release of prostaglandin E2 from cultured amnion cells, suggesting a possible role of inhibin and activin in fetal membranes

Attenuation of kindled seizures by intranasal delivery of neuropeptide-loaded nanoparticles.

Thyrotropin-releasing hormone (TRH; Protirelin), an endogenous neuropeptide, is known to have anticonvulsant effects in animal seizure models and certain intractable epileptic patients. Its duration of action, however, is limited by rapid tissue metabolism and the blood—brain barrier. Direct nose-to-brain delivery of neuropeptides in sustained-release biodegradable nanoparticles (NPs) is a promising mode of therapy for enhancing CNS neuropeptide bioavailability. To provide proof of principle for this delivery approach, we used the kindling model of temporal lobe epilepsy to show that 1) TRH-loaded copolymer microdisks implanted in a seizure focus can attenuate kindling development in terms of behavioral stage, after-discharge duration (ADD), and clonus duration; 2) intranasal administration of an unprotected TRH analog can acutely suppress fully kindled seizures in a concentration-dependent manner in terms of ADD and seizure stage; and 3) intranasal administration of polylactide nanoparticles (PLA-NPs) containing TRH (TRH-NPs) can impede kindling development in terms of behavioral stage, ADD, and clonus duration. Additionally, we used intranasal delivery of fluorescent dye-loaded PLA-NPs in rats and application of dye-loaded or dye-attached NPs to cortical neurons in culture to demonstrate NP uptake and distribution over time in vivo and in vitro respectively. Also, a nanoparticle immunostaining method was developed as a procedure for directly visualizing the tissue level and distribution of neuropeptide-loaded nanoparticles. Collectively, the data provide proof of concept for intranasal delivery of TRH-NPs as a viable means to 1) suppress seizures and perhaps epileptogenesis and 2) become the lead compound for intranasal anticonvulsant nanoparticle therapeutics