Neuregulin-2 ablation results in dopamine dysregulation and severe behavioral phenotypes relevant to psychiatric disorders.

Numerous genetic and functional studies implicate variants of Neuregulin-1 (NRG1) and its neuronal receptor ErbB4 in schizophrenia and many of its endophenotypes. Although the neurophysiological and behavioral phenotypes of NRG1 mutant mice have been investigated extensively, practically nothing is known about the function of NRG2, the closest NRG1 homolog. We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior in the elevated plus maze and deficits in the T-maze alteration reward test-a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with N-methyl-d-aspartate receptor (NMDAR) antagonists, we demonstrate that NMDAR synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDARs. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders.

ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors.

Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies have focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit different regional imbalances of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum measured by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4f/f mice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that direct NRG/ErbB4 signaling in DAergic axonal projections modulates DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors relevant to psychiatric disorders.

Regulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons.

Neuregulin (NRG)/ErbB receptor signaling pathways have recently been implicated in the reversal of long-term potentiation at hippocampal glutamatergic synapses. Moreover, polymorphisms in NRG-1 and ErbB-4 genes have been linked to an increased risk for developing schizophrenia. ErbB-4 is highly expressed at glutamatergic synapses where it binds to PSD-95 via its carboxyl terminal T-V-V sequence. Here we investigated the expression, localization and trafficking of ErbB-4 in cultured hippocampal neurons by immunocytochemistry, surface protein biotinylation, and live labeling of native receptors. We show that neuronal ErbB-4 is detected at its highest levels in GABAergic interneurons, as observed in vivo. ErbB-4 immunoreactivity precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95-positive puncta. By surface protein biotinylation, the fraction of ErbB-4 receptors on the plasma membrane increases from 30% to 65% between 6 and 16 days in vitro (DIV). Interestingly, 30 min of NRG stimulation triggers measurable ErbB-4 receptor internalization at DIV 16, despite increased colocalization with PSD-95. We also investigated the role of TNFalpha-converting enzyme (TACE)-mediated receptor processing in regulating ErbB-4 surface expression. We found that the cleavage-resistant JM-b isoform accounts for 80% of all ErbB-4 transcripts in cultured hippocampal neurons. Receptor stimulation or treatment with phorbol esters does not induce detectable ErbB-4 processing, indicating that neurons mostly rely on endocytosis of the intact receptor to regulate ErbB-4 surface expression. These results enhance our understanding of the regulation of ErbB-4--mediated signaling at glutamatergic synapses.

[PHS Repair in femoral hernia surgery]. / La chirurgia dell'ernia femorale con sistema PHS.

BACKGROUND: Since December 2000 we began to treat femoral hernias, for the first time in emergency, placing the underlay mesh of a PHS 168; device in the properitoneal space using the same anterior way well known in plug technique repair. The technical description and short term effectiveness, safeness and comfort of our new femoral prosthetic repair are reported. METHODS: Prospective analysis of 12 unselected consecutive female patients who underwent 12 PHS device femoral hernia repairs (6 in emergency with 2 intestinal resections and 6 in elective settings), performed from December 2000 to December 2001 at the Institute of General Surgery, University of Ferrara. Mean age was 61,7 (range 25-94) and mean follow-up was 6 months (from 1 to 11). RESULTS: Mean hospital stay was 2.1 days (from 1 to 5). No recurrences or complications occurred. We report one (8.3%) 25 year old woman with moderate cruralgia which required analgesics assumption form more then 24 hours. CONCLUSIONS: Although our report is not statistical significant (recent introduction of this new technique for a low incidence rate pathology) actually we perform PHS device femoral hernia repair systematically, both in elective and emergency surgery. This new procedure appears as a simple, safe, effectiveness and comfortable, anterior properitoneal "tension-free" repair of the myopectineal region (direct inguinal hernias prophylaxis).

[Neuregulins: A family of factors with critical functions during nervous system development and in the cellular transformation and differentiation]. / Las neuregulinas: una familia de factores con funciones críticas en el desarrollo del sistema nervioso y en la diferenciación y transformación celular.

INTRODUCTION: The neuregulins are a family of factors that perform important functions during the development of the nervous system, neuromuscular junction and heart. These factors are also involved in nervous system disorders, and in the generation and progression of tumors. DEVELOPMENT: There are four genes and many isoforms which share a similar molecular structure. The neuregulins are differentially expressed and bind with different affinities to specific combinations of ErbB receptors. This level of complexity is directing numerous researching in order to understand the molecular mechanisms that regulate a specific pathway. CONCLUSION: Neuregulin ErbB receptor complexes activate specific intracellular pathways that culminate in very different fates: differentiation or proliferation.

[Inguinal hernia prosthetic repair through the anterior approach]. / La riparazione protesica elettiva dell'ernia inguinale per via anteriore.

BACKGROUND: Aim of this study is to compare the Lichtenstein's, Rutkow's and PHS techniques of inguinal hernia repair in terms of therapeutical efficacy and grade of acceptability, expressed in function of the complications, compliance and performance status of the patients. METHODS: The preliminary results of an ongoing prospective non-randomized study on the most frequently used techniques of inguinal hernia repair (PHS, Rutkow's, and Lichtenstein's) are reported. Sixty patients with primary inguinal hernia were divided into three homogeneous groups for age, gender, Gilbert's type of hernia, type of anesthesia, ASA class. The three groups underwent PHS, Rutkow's and Lichtenstein's inguinal hernia repairs, respectively. The end-points of the study were: operative time, intra- and postoperative pain, intra- and postoperative complications, patients compliance and performance status. RESULTS: The mean operative time were 40', 41' and 36' minutes for the PHS, Rutkow's and Lichtenstein's procedures, respectively. One of patients of the PHS group, five of the Rutkow's and none of those undergoing Lichtenstein's repair needed mild intraoperative sedation. Mild postoperative pain was recorded in 5% of the patients undergoing PHS repair and 10% undergoing Rutkow's repair. No intraoperative complications, difference in compliance and performance status were detected in the three groups. CONCLUSIONS: The conclusion is drawn that the PHS, Rutkow's and Lichtenstein's procedures for inguinal hernia repair are safe (no complications), effective and well accepted by the patients (85% of the patients expressed a very good judgement) although the Rutkow's repair seems more invasive. The appearance of a better trend, in patient's compliance and performance status when operated with the PHS technique, need to be confirmed in the future but, if it will be, this could became our first choice technique of repair for the medium and large hernia defect.

Neuregulin and ErbB receptor signaling pathways in the nervous system.

The neuregulins are a complex family of factors that perform many functions during neural development. Recent experiments have shown that neuregulins promote neuronal migration and differentiation, and regulate the selective expression of neurotransmitter receptors in neurons and at the neuromuscular junction. They also regulate glial commitment, proliferation, survival and differentiation. At interneuronal synapses, neuregulin ErbB receptors associate with PDZ-domain proteins at postsynaptic densities where they can modulate synaptic plasticity. How this combinatorial network - comprising many neuregulin ligands that signal through distinct combinations of dimeric ErbB receptors - elicits its multitude of biological effects is beginning to be resolved.

ErbB transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat central nervous system.

The neuregulin (NRG) family of growth and differentiation factors and their erbB receptors contribute importantly to the development of the nervous system, but their distribution and function in the adult brain are poorly understood. The present study showed that erbB2, erbB3, and erbB4 transcripts and protein are distributed throughout all areas of adult rat brain. These three receptors were differentially expressed in neurons and glia. Some neurons expressed only a subset of erbB kinases, whereas other neurons expressed all three erbB receptors but sequestered each of these polypeptides into distinct cellular compartments. In synapse-rich regions, erbB immunoreactivity appeared as punctate-, axon-, and/or dendrite-associated staining, suggesting that NRGs are involved in the formation and maintenance of synapses in adult brain. ErbB labeling also was present in neuronal soma, indicating that NRGs act at sites in addition to the synapse. Glia in adult brain also differentially expressed erbB3 and erbB4. Approximately half of the erbB3 labeling in white matter was associated with S100beta+/glial fibrillary acidic protein negative macroglia (i.e., oligodendrocytes or glial fibrillary acidic protein negative astrocytes). In contrast, macroglia in gray matter did not express erbB3. The remaining erbB3 immunoreactivity in white matter and erbB4 glial staining seemed to be associated with microglia. These results showed that erbB receptors are expressed widely in adult rat brain and that each erbB receptor subtype has a distinct distribution. The differential distributions of erbB receptors in neurons and glia and the known functional differences between these kinases suggest that NRGs have distinct effects on these cells. The continued expression of NRGs and their erbB receptors in mature brain also implies that these molecules perform important functions in the brain throughout life.

Neuregulin found in cultured-sciatic nerve conditioned medium causes neuronal differentiation of PC12 cells.

The present work deals with the search and identification of the molecule or combination of molecules, present in a medium conditioned by cultured rat-sciatic nerves (CM), able to cause neuronal differentiation of PC12 cells. The molecular mass range of the active fraction, as well as the thermostability and heparin affinity of the active component found in previous work, all characteristics shared with neuregulin (NRG) family members, led us to search for a NRG protein in the CM. Nerves were previously cultured for 8 days and the CM collected every 24 h, the following 3 days. The CM was concentrated (30,000 NMWL) and fractionated by quaternary ammonium chromatography and Cibacron blue affinity chromatography. The most active fraction B1.2 was further characterized by heparin affinity chromatography, size exclusion HPLC, Western blotting and immunoprecipitation. Results reveal abundance of NRG mRNA in the cultured nerves, presence of a 54 kDa NRG protein in the CM that increases along fractionation, and progressive diminution of fraction B1.2 differentiation activity on PC12 cells by gradual removal of the NRG protein by immunoprecipitation. The abundance of Schwann cells and the lack of axons in the cultured nerves suggest Schwann cells as the main NRG source, to which fibroblasts and perineurial cells might contribute.

The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses.

Neuregulins regulate the expression of ligand- and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1-4. The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown. We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD). The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain. Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95/SAP90, PSD-93/chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with beta(2)-syntrophin, which has a single PDZ domain. As with N-methyl-D-aspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95. Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates. Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons. ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals. The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals. As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity.

Neuregulin-beta induces expression of an NMDA-receptor subunit.

Neuregulins (also known as ARIA, NDF, heregulin, GGF) are a family of widely expressed growth and differentiation factors. Neuregulins secreted from motor neurons accumulate at maturing neuromuscular junctions, where they stimulate transcription of genes encoding specific acetylcholine receptors. How these factors function at central synapses, however, is unknown. In the maturing cerebellum, neuregulins are concentrated in glutamatergic mossy fibres that innervate granule cells in the internal granule-cell layer. We have analysed the effects of neuregulins on the expression of genes encoding NMDA (N-methyl-D-aspartate) receptors in the cerebellum, because receptor composition changes dramatically as expression of the receptor NR2C subunit is specifically induced in neurons in the internal granule-cell layer during synaptogenesis. Here we report that addition of a neuregulin-beta isoform to cultured cerebellar slices specifically increases the expression of NR2C messenger RNAs by at least 100-fold; effects are only minor with a neuregulin-alpha isoform. This stimulation of NR2C expression requires synaptic activity by NMDA receptors, as well as neuregulin-beta. Addition of the NMDA-receptor-channel blocker AP-5 prevents upregulation of the NR2C subunit by neuregulin, whereas an AMPA/kainate-receptor antagonist does not. Consistent with these effects of neuregulin, we find that granule cells express its receptors ErbB2 and ErbB4 before the NR2C subunit of the NMDA receptor. Our results indicate that neuregulins regulate the composition of neurotransmitter receptors in maturing synapses in the brain, in a manner analogous to the neuromuscular junction.

Isolation and structure of the rat gene encoding troponin I(slow).

Troponin I is a myofibrillar protein involved in the Ca(2+)-mediated regulation of actomyosin ATPase activity. We report here the isolation and characterization of the gene coding for the slow-muscle-specific isoform of the rat troponin I polypeptide (TpnI). Using restriction mapping, PCR mapping and partial DNA sequencing, we have determined the exon/intron arrangement of this gene. The transcription unit is 10.5-kb long and contains nine exons ranging in size from 4 bp to 330 bp. The rat TpnI(slow) gene is interrupted by large intervening sequences; a 3.3-kb intron separates the 5' untranslated exons from the protein-coding exons. Comparison of the structure of rat TpnI(slow) with that of quail TpnI(fast) reveals that they have a similar intron/exon organization. The 5' untranslated region of the rat gene contains an additional exon, otherwise, the positions of introns and coding exons map to essentially identical regions in both genes.

Changes in the expression of mRNAs for myogenic factors and other muscle-specific proteins in experimental autoimmune myasthenia gravis.

The regulation of genes for acetylcholine receptor (AChR), myogenic factors and other muscle-specific proteins has been analyzed in experimental autoimmune myasthenia gravis (EAMG) and following denervation. The levels of the transcripts for the myogenic factors, MyoD1, myogenin and MRF4, were measured using Northern blot analysis. Myogenin and MRF4 transcript levels were observed to be 3.1- and 2.6-fold higher in muscle of rats with EAMG than in controls, respectively. MyoD1 levels, however, remained unchanged. The increases in AChR, myogenin and MRF4 mRNAs were one order of magnitude higher in 2-week denervated muscle than in the myasthenic muscle. The levels of muscle creatine kinase (MCK), alpha-actin and muscle dystrophin transcripts were also analyzed. Dystrophin levels were found to be 1.7- and 4.7-fold higher in EAMG and denervated muscle, respectively, than in controls; in contrast, MCK and alpha-actin levels remained unchanged.

Isolation and characterization of the beta and epsilon subunit genes of mouse muscle acetylcholine receptor.

The genes coding for the beta and epsilon subunits of the mouse muscle nicotinic acetylcholine receptor (nAChR) were mapped by Southern blot analysis, and the entire loci for both genes cloned. The results indicate that they are single-copy genes. Both were sequenced to determine their size and structural organization. The beta subunit gene spans approximately 8 kilobases and is organized into 11 exons. A region containing cysteines, which are thought to form a disulfide bond and which are highly conserved, is encoded by one exon in all muscle acetylcholine receptor genes with the exception of the beta subunit gene, where it is split into two exons. The epsilon subunit gene spans 4.3 kilobases and contains 12 exons; it has the same structure as the gamma and delta nAChR genes. The intron-exon boundaries and exonic organization of the five known nAChR genes were compared. The analysis showed that the first 4 exons and the last exon of all muscle and brain nAChR subunit genes have the same boundaries, with the exception of a nAChR-related gene in Drosophila.

Clinical and diagnostic features of portosystemic shunt in a foal.

Portosystemic shunt was diagnosed in a 6-month-old Quarter Horse filly with acute onset of apparent blindness and a 3-month history of depression, lethargy, and ataxia. Clinicopathologic test results indicated slightly high gamma-glutamyl transpeptidase activity and serum total bilirubin concentration. Sulfobromophthalein half time was prolonged, and plasma ammonia and serum bile acid concentrations were high as well. Histopathologic findings of percutaneous liver biopsy included widespread hepatocyte atrophy and numerous prominent small arterioles in the area of the portal triad. On the basis of history, clinical findings, and clinicopathologic abnormalities, a presumptive diagnosis of portosystemic vascular anomaly was made. To confirm the tentative diagnosis, nuclear hepatic scintigraphy and operative mesenteric portography were performed. Medical treatment was unsuccessful, and the foal was euthanatized. Portosystemic shunts have been described in dogs and cats, but few cases have been reported in large animal species. Other, more common causes of neurologic abnormalities in foals, such as trauma, vertebral body abscesses, brain abscesses, and meningitis, must be ruled out before portosystemic shunt is considered.

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit.

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.

Sodium channel activity in brain membrane fractions isolated from rats of different ages.

The Na+ channel activity (tetrodotoxin sensitive 22Na+ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79-87) from rats 5, 10, 30 and 60 days old. The 22Na+ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 microM) increases the 22Na+ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 microM) causes an increment of the 22Na+ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 microM), the K0.5 for veratridine is diminished and the maximum 22Na+ flux is increased. The increments of 22Na+ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin (K0.5 approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na+ channel activity. The other subfraction is enriched in myelin and shows no Na+ channel activity. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na+ channel activity.