The Iron Man of the Renaissance: the contribution of Girolamo Fabrizi d'Acquapendente.

PURPOSE: To highlight the most relevant contributions of the Italian Renaissance surgeon Girolamo Fabrizi d'Acquapendente in the field of orthopaedics and traumatology. METHODS: An extensive research on the life and achievements of Girolamo Fabrizi was conducted on University Libraries as well as on electronic databases like PubMed. RESULTS: Girolamo Fabrizi d'Acquapendente is known for his embryology and physiology studies, particularly on sensory organs and blood circulation. He founded the world's first permanent anatomical theater established at the University of Padua and inaugurated in 1595. His most notable publications include surgical and definitely orthopedics works such as "De fracturis" (On fractures) and "De luxationibus" (On joint displacement). He outlined some principles of treatment that are still valid nowadays such as anatomical reduction and stabilization of the fracture, that were applied using the equipment available at that time. He described and illustrated maneuvers and instruments, such as pulleys, winches, splint, and bandages. He further depicted the famous "Oplomochlion" ("the armored man"), which is actually a collection of all of the braces used at the time to correct congenital and post-traumatic deformities. CONCLUSION: Bracing and prosthetic replacements have accompanied medical history throughout the centuries, from Ancient Egypt to the present, but it was the ingenuity of Renaissance surgeons that pushed biomedical technology to new heights: Girolamo Fabrizi d'Acquapendente was one of the most illustrious contributors to these great achievements.

Combined toxic effects and DNA damage to two plant species exposed to binary metal mixtures (Cd/Pb).

Acute and long-term (3-, 10- and 56-day exposure) laboratory toxicity tests were carried out to assess the individual and combined toxic effects of cadmium (Cd) and lead (Pb) in Brassica oleracea and Trifolium repens. In addition to morphological parameters, this work also used comet assay to address endpoints in relation to genotoxicity. Bioaccumulation was measured to demonstrate the influence of the mixture on the concentrations of each metal in the plant. The statistical method reported by Ince et al. (1999) was used to evaluate the types of interaction between Cd and Pb in each treatment and concerning their combined effect. This study concludes that the combined effects of binary metal combinations of Cd/Pb on morphological parameters are most often additive, sometimes antagonistic and more rarely synergistic, thus extending the findings of previous publications on this subject. DNA damage analysis revealed concentration- and time-dependent interactions. Synergistic effects of mixed metals (more breaks than individually applied metals) are observed in T. repens after a short exposure. Antagonistic effects are statistically significant after 10 days-exposure, suggesting competition between metals. At 56 days, the rate of DNA damage observed in plants exposed to the Cd/Pb mixture was similar to that measured in plants exposed to lead only and was significantly lower than the rate of DNA damage induced by Cd. This supports the idea that there may be competition between metals and also strengthens the hypothesis that long-term reparation mechanisms may be implemented. Cd/Pb co-exposure does not significantly influence the bioaccumulation of each metal. It is nevertheless important to note that a statistically significant 'interaction' is not necessarily biologically relevant and should therefore be considered with caution when assessing heavy metals combined effects.

Antioxidant responses of Annelids, Brassicaceae and Fabaceae to pollutants: a review.

Pollutants, such as Metal Trace Elements (MTEs) and organic compounds (polycyclic aromatic hydrocarbons, pesticides), can impact DNA structure of living organisms and thus generate damage. For instance, cadmium is a well-known genotoxic and mechanisms explaining its clastogenicity are mainly indirect: inhibition of DNA repair mechanisms and/or induction of Reactive Oxygen Species (ROS). Animal or vegetal cells use antioxidant defense systems to protect themselves against ROS produced during oxidative stress. Because tolerance of organisms depends, at least partially, on their ability to cope with ROS, the mechanisms of production and management of ROS were investigated a lot in Ecotoxicology as markers of biotic and abiotic stress. This was mainly done through the measurement of enzyme activities The present Review focuses on 3 test species living in close contact with soil that are often used in soil ecotoxicology: the worm Eisenia fetida, and two plant species, Trifolium repens (white clover) and Brassica oleracea (cabbage). E. fetida is a soil-dwelling organism commonly used for biomonitoring. T. repens is a symbiotic plant species which forms root nodule with soil bacteria, while B. oleracea is a non-symbiotic plant. In literature, some oxidative stress enzyme activities have already been measured in those species but such analyses do not allow distinction between individual enzyme involvements in oxidative stress. Gene expression studies would allow this distinction at the transcriptomic level. A literature review and a data search in molecular database were carried out on the basis of keywords in Scopus, in PubMed and in Genbank™ for each species. Molecular data regarding E. fetida were already available in databases, but a lack of data regarding oxidative stress related genes was observed for T. repens and B. oleracea. By exploiting the conservation observed between species and using molecular biology techniques, we partially cloned missing candidates involved in oxidative stress and in metal detoxification in E. fetida, T. repens and B. oleracea.

External fixator-assisted plating osteosynthesis in a rabbit model of femoral bone defects appears to be a feasible and reproducible surgical technique: preliminary insights from a bone substitute study.

PURPOSE: The aims of this study were to assess the complications associated with the use of an external fixator-assisted plate osteosynthesis technique to stabilize a femoral bone defect in a rabbit model and to evaluate if this technique could avoid the mispositioning and the displacement of the femoral fragments during the surgical procedure. METHODS: A preliminary cadaveric animal study was conducted to develop a new technique of external fixator-assisted plating. Thirty rabbits underwent a surgical procedure consisting in the creation of a femoral bone defect and, subsequently an implantation of a bone substitute through the assistance of a temporary external fixator. The fixator's ability to maintain length and alignment during surgery was documented. All intraoperative complications were prospectively collected. RESULTS: No complications related to the use of the temporary external fixator were reported. The technique successfully prevented mispositioning and dislocation during plating in all the rabbits. CONCLUSION: In a rabbit animal model, the use of an external fixator-assisted plate osteosynthesis technique appears to be feasible and effective in avoiding misposition and rotation of femoral fragments when performing osteotomy and plating to create a mid-diaphyseal femoral defect.

Biomarker measurements in Trifolium repens and Eisenia fetida to assess the toxicity of soil contaminated with landfill leachate: a microcosm study.

To assess the toxicity of a soil contaminated with landfill leachate, biomarker measurements in two species living in close contact with the soil, i.e. a plant species Trifolium repens and an animal species Eisenia fetida, were conducted. Briefly, both species were studied after simultaneous exposure conducted in microcosms. The organisms were exposed to soil supplemented with pure leachate, leachate diluted to 50%; leachate diluted to 25% and without leachate. After a 10 weeks exposure period, we observed an increase in the Olive Trail Moment in T. repens, compared to the reference, for 50% and pure leachate. The response observed appears to be dose-dependent and linear in our experimental conditions. Addition of the leachate to the reference soil induced an increase in Cd-Metallothionein-coding mRNA quantity in E. fetida. In addition, expression level of another gene implied in detoxification and coding Phytochelatin synthase was significantly induced in worms exposed to the reference soil spiked with the leachate, regardless presence of T. repens. Thus, T. repens and E. fetida can be used in a complementary manner to assess soil quality. Sensitivities of the test species yield sensitive bioassays as both species responded at low doses despite the buffering effect of the soil.

Minimally Manipulated Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis: A Systematic Review of Clinical Evidence.

BACKGROUND: The use of laboratory-expanded mesenchymal stem cells (MSCs) is subject to several restrictions, resulting in "minimal manipulation" methods becoming the current most popular strategy to increase the use of MSCs in an orthopaedic practice. The aim of the present systematic review is to assess the clinical applications of "minimally" manipulated MSCs, either as bone marrow aspirate concentrate (BMAC) or as stromal vascular fraction (SVF), in the treatment of knee osteoarthritis (OA). METHODS: A systematic review of three databases (PubMed, ScienceDirect, and Google Scholar) was performed using the following keywords: "Knee Osteoarthritis" with "(Bone marrow aspirate) OR (bone marrow concentrate)" or with "(adipose-derived mesenchymal stem cells) OR (adipose derived stromal cells) OR (stromal vascular fraction) OR (SVF)" as either keywords or MeSH terms. The reference lists of all retrieved articles were further reviewed for identification of potentially relevant studies. RESULTS: Twenty-three papers were included in the final analysis (10 on BMAC and 13 on SVF). Of these, only 4 were randomized controlled trials (RCTs). Bias risk evaluation, performed using a modified Coleman score, revealed an overall poor quality of the studies. In terms of clinical application, despite the apparent safety of minimally manipulated MSCs and the short-term positive clinical outcomes associated with their use, clinicians reported different preparation and administration methods, ranging from single intra-articular injections to intraosseous applications to administration in combination with other surgical procedures. CONCLUSIONS: The available literature is undermined by both the lack of high-quality studies and the varied clinical settings and different protocols reported in the few RCTs presently published. This prevents any recommendation on the use of either product in a clinical practice. Nevertheless, the use of minimally manipulated MSCs (in the form of BMAC or SVF) has been shown to be safe and have some short-term beneficial effects.

Antioxidant defense gene analysis in Brassica oleracea and Trifolium repens exposed to Cd and/or Pb.

This study focused on the expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10, and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (glutathione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand the detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase upregulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, glutathione transferase, and peroxidase upregulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.

Combined effect of Cd and Pb spiked field soils on bioaccumulation, DNA damage, and peroxidase activities in Trifolium repens.

The present study was designed to investigate the combined effects of Cd and Pb on accumulation and genotoxic potential in white clover (Trifolium repens). For this purpose, T. repens was exposed to contaminated soils (2.5-20 mg kg(-1) cadmium (Cd), 250-2000 mg kg(-1) lead (Pb) and a mixture of these two heavy metals) for 3, 10 and 56 days. The resulting bioaccumulation of Cd and Pb, DNA damage (comet assay) and peroxidase activities (APOX and GPOX) were determined. The exposure time is a determinant factor in experiments designed to measure the influence of heavy metal contamination. The accumulation of Cd or Pb resulting from exposure to the two-metal mixture does not appear to depend significantly on whether the white clover is exposed to soil containing one heavy metal or both. However, when T. repens is exposed to a Cd/Pb mixture, the percentage of DNA damage is lower than when the plant is exposed to monometallic Cd. DNA damage is close to that observed in the case of monometallic Pb exposure. Peroxidase activity cannot be associated with DNA damage under these experimental conditions.

Distribution, cellular localization, and ontogeny of preprothyrotropin-releasing hormone-(160-169) (Ps4)-binding sites in the rat pituitary.

The rat TRH precursor contains five copies of TRH separated by connecting peptides. Previous studies have shown that the decapeptide prepro-TRH (160-169; Ps4) potentiates the effect of TRH on TSH secretion. In the present study, we have characterized Ps4 receptors in the rat pituitary by in vitro autoradiography using [125I-Tyr0]Ps4 as a radioligand, and we have investigated the evolution of receptor density during ontogenesis. Incubation of rat pituitary slices with [125I-Tyr0]Ps4 revealed intense binding in the anterior lobe and virtually no binding in the neurointermediate lobe. Biochemical characterization of the Ps4-binding sites suggested the existence of a single class of sites exhibiting high affinity for [Tyr0]Ps4 (IC50 = 8.3 +/- 1.2 nM) and a much lower affinity for Ps4 (IC50 = 9.3 +/- 1.2 microM). Emulsion-coated cytoautoradiography performed on cultured anterior pituitary cells showed that only 26% of the cells possessed [125I-Tyr0]Ps4-binding sites. Immunocytochemical analysis using antibodies against the different anterior pituitary hormones indicated that the cells possessing [125I-Tyr0]Ps4-binding sites did not correspond to TSH-, PRL-, GH-, ACTH-, or LH-secreting cells. In contrast, cells expressing Ps4 receptors were immunoreactive for the S-100 protein, a marker of folliculo-stellate cells. During postnatal development, a 4-fold increase in the concentration of [125I-Tyr0]Ps4-binding sites occurred from birth to weaning in the pituitary, with a marked and transient increase at the time of weaning. Thereafter, the density of sites declined gradually until day 60. In conclusion, the present study shows that folliculo-stellate cells express [125I-Tyr0]Ps4-binding sites in the anterior pituitary, and that these sites are developmentally regulated. The present data suggest that the potentiating effect of Ps4 on TRH-induced TSH secretion is mediated by folliculo-stellate cells.

Original involvement of antimicrobial peptides in mussel innate immunity.

Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti-infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects.

Structural characterization of osmoregulator peptides from the brain of the leech Theromyzon tessulatum: IPEPYVWD and IPEPYVWD-amide.

Neurons immunoreactive to an antiserum (a-OT) directed specifically against the C-terminal part (prolyl-leucyl-glycinamide) of vertebrate oxytocin (OT) were detected in the brain of the leech Theromyzon tessulatum. With high pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts, evidence was given of the presence of three peptides (P1, P2, P3) immunoreactive to a-OT. Results of injection experiments in T. tessulatum and of titrations of each peptide at the different physiological stages of the animals which showed a peak in peptide P1 amount at stage 3B, indicated that P1 is the active OT-like peptide. Using three steps of reversed-phase HPLC, Edman degradation and electrospray mass spectrometry, two sequences for P1 (IPEPYVWD and IPEPYVWD-amide) were found. These peptides differ from peptides to the oxytocin/vasopressin family and are unique in the animal kingdom. Confirmation of their action on the hydric balance and their distribution in the CNS were presented.

Leech egg-laying-like hormone: structure, neuronal distribution and phylogeny.

Cells immunoreactive to antisera specifically directed against Lymnaea stagnalis caudo dorsal cells egg-laying hormone (CDCH) or against alpha- and beta-peptides (CDCP), encoded on the egg-laying hormone precursor, were detected in central nervous system (CNS) of the rhynchobdellid leech Theromyzon tessulatum. A co-localization of the CDC-like hormone and CDC-like peptides was found in T. tessulatum as in L. stagnalis CNS. approximately 45 immunoreactive cells to the anti-CDCH were detected in leech brain but this number varies according to the stage of the animal life cycle, i.e. it reaches a maximum just before egg-laying while after it decreases to 2-3 cells. CDCH and alpha-CDCP epitopes recognized by anti-CDCH and anti-alpha-CDCP were contained in neurosecretory granules. Following an extensive purification, including HPGPC and reverse-phase HPLC, the CDC-like hormone contained in the T. tessulatum CNA was isolated. The sequence (GSGVSNGGTEMIQLSHIRERQRYWAQDNLRRRFLEK-amide) was established by a combination of automated Edman degradation, arginyl-endopeptidase digestion, electrospray mass spectrometry measurement and carboxypeptidase A treatment. The results demonstrate that the peptide recognized by the anti-CDCH in the leech CNS possesses 27.8, 37.2 and 47.2% sequence identity with Aplysia parvula, Lymnaea stagnalis and Aplysia californica ELH, respectively. This molecule was named the leech egg-laying-like hormone (L-ELH). The secondary structure prediction of the L-ELH and all mollusks ELH, revealed the existence of a conserved segment (segment 29-34) in a strong helicoidal bend that might be important for receptor recognition and/or activation. This finding constitutes the first biochemical characterization of an egg-laying hormone in other invertebrates than mollusks.

Biochemical identification and ganglionic localization of leech angiotensin-converting enzymes.

We demonstrate the presence of a membrane and soluble form of leech Theromyzon tessulatum angiotensin-converting enzyme (ACE). Four steps in the purification of this enzyme include gel-permeation, captopril-sepharose affinity and anion-exchange chromatography followed by a reverse-phase HPLC. The peptidyl dipeptidases (of approximately 120 and approximately 100 kDa) are glycosylated enzymes hydrolysing the Phe8-His9 bond of angiotensin I, exhibiting the same specific activity and Km whereas the soluble ACE exhibits a higher catalytic efficiency. This hydrolysis is inhibited by the ACE-specific antagonist captopril. Western blot analysis of a polyclonal antiserum raised against the first 11 amino-acid residues of the membrane ACE and the N-terminal sequence of the soluble molecule also demonstrates the presence of two ACE enzymes. Anti-ACE immunocytochemistry also supports the presence of two forms of ACE. This material is found in neurons and glia. We demonstrate for the first time the cellular localization and biochemical characterization of ACEs in the central nervous system of an invertebrate. Thus, the leech brain may represent a simple model for the study of these enzymes.

Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis.

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.

Detection of neutral endopeptidase (NEP, enkephalinase, E.C.3.4.24.11) in relation to dopaminergic and gonadoliberinergic nerve endings in the median eminence of the male rat: a double labeling ultrastructural study.

The existence of neutral endopeptidase (Enkephalinase, NEP, E.C.3.4.24.11) in membranes of nerve endings in the rat median eminence suggests that some neuropeptides have paracrine and/or autocrine actions in this region. In vitro, neutral endopeptidase is capable of hydrolysing a variety of regulatory peptides but in vivo, many works indicate that in the central nervous system this enzyme is highly implicated in the biological inactivation of enkephalins and tachykinins. In addition there is evidence that NEP is also involved in the inactivation of neurotensin in vivo. The modulation of the release of gonadotrophin releasing hormone (GnRH) is one of the documented actions of enkephalins within the median eminence. However, it is at present unclear whether enkephalins act on dopamine endings, on GnRH endings or on both. As the technical parameters and particularly the tissue fixation used to detect neutral endopeptidase are compatible with immunocytochemical detection of GnRH and tyrosine-hydroxylase (the rate limiting enzyme in the synthesis of catecholamines), two double immunolabelings were realised at the ultrastructural level to determine if GnRH and dopamine nerve endings have the enzyme inserted within their plasma membrane. Our study shows the presence of neutral endopeptidase on tyrosine-hydroxylase-immunoreactive nerve endings while presence of the enzyme on GnRH-immunoreactive nerve endings is not demonstrated. Consequently, our results provide morphological arguments for possibilities of paracrine and/or autocrine actions by neuropeptides inactivated by neutral endopeptidase on tuberoinfundibular dopaminergic nerve endings. Conversely, action of the same peptides on GnRH boutons seems more unlikely.

Radioimmunocytochemical distribution of neutral endopeptidase (enkephalinase E.C.3.4.24.11) at the ultrastructural level in the rat median eminence.

Neutral endopeptidase (E.C.3.4.24.11) was visualized at the ultrastructural level in the external zone of the rat median eminence by using 125I-labelled IgG of a monoclonal serum. A precise analysis of the localization of the immunolabelling, which appears in the form of individual stray silver grains, was undertaken. Among the 1,045 grains counted, 82% were localized over membrane appositions involving nerve endings only and nerve endings plus tanycytes. The difference between the real and a randomly generated population of grains was statistically significant. Our results provide morphological arguments in support of the view of a paracrine action of neuropeptides present in the median eminence especially enkephalins but possibly, substance P, angiotensin, cholecystokinin and neurotensin. These neuropeptides are known to be inactivated by neutral endopeptidase. The action of these peptides may be exerted on nerve endings (autocrine or paracrine) but an intervention on tanycytes cannot be excluded.

Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.

Concomitant variation in immunoreactivity for enkephalins and neutral endopeptidase in the median eminence of the lactating rat.

In the brain, neutral endopeptidase (NEP) is implicated in the biological inactivation of enkephalins and substance P. NEP, enkephalins and neurokinins were detected by immunocytochemistry in the median eminence (ME) of lactating female rats either freely nursing or separated from their pups for 48 h. A significant decrease in enkephalin and NEP immunoreactivity was observed in the external zone of the ME after pup removal. In both animal groups, neurokinin immunoreactivity was absent from this region. These results indicate that: (1) a concomitant variation in immunoreactivity for enkephalins and NEP occur in the ME of the lactating rat; (2) in the ME during lactation, NEP may essentially be implicated in the biological degradation of enkephalins.

A reappraisal of pacemaker timing cycles pertaining to automatic mode switching.

Automatic mode switching algorithms of dual chamber pacemakers require fundamental changes in the operation of pacemaker timing cycles to optimize detection of supraventricular tachyarrhythmias. The timing cycles related to mode switching are basically independent of the algorithm design. Blanking periods (when the sensing amplifier is temporarily disabled) should be optimized to a relatively small fraction of the pacing cycle to enhance atrial sensing and prevent far-field sensing. This review explains the function of the timing cycles pertaining to mode switching and proposes simpler terminology to facilitate the understanding of pacemaker function and electrographic interpretation of complex recordings.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy.

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.