[Pediatric ADHD: what does the otolaryngologist need to know?]. / ADHS im Kindesalter: Was muss der HNO-Arzt wissen?

According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria, attention deficit hyperactivity disorder (ADHD) affects around 5% of all children and adolescents worldwide. The causes of ADHD are multifactorial, with a large genetic influence but also involvement of exogenic and psychosocial factors. Its core symptoms consist of attention deficits, hyperactivity and disruption of impulse control. It is important that symptoms appear before the age of six and are evident in multiple different situations, such as in familial and school environments. ADHD is a dimensional disorder, which means that the diagnostic process is time consuming, comprising a physical and neurological examination, behavioral observations and differentiated psychological assessments. In the field of otolaryngology, ADHD represents one of the important differential diagnoses to an auditory processing disorder (APD), alongside reading- and writing impairments and delayed speech development. In the instance of additional behavioral problems or more severe symptoms, it is advisable to transfer the patient to a specialized pediatrician or child and adolescent psychiatrist for appropriate counseling and treatment where required.

Gene transfer through the blood-nerve barrier: NGF-engineered neuritogenic T lymphocytes attenuate experimental autoimmune neuritis.

Nerve-specific autoimmune T lymphocytes were used as vehicles to deliver therapeutically useful neurotrophic factors across the endothelial blood-nerve barrier. P2 protein-reactive T-lymphocyte lines from Lewis rats were transduced with a recombinant retrovirus containing the mouse nerve growth factor (NGF) gene. The engineered T cells released high amounts of NGF dependent on antigenic stimulation in vitro. After intravenous injection, the T cells infiltrated the rat peripheral nervous system and persisted there for at least two weeks. Local release of NGF from engineered T cells was demonstrable by immunocytochemistry and by an anti-inflammatory effect on infiltrating macrophages.

Experimental autoimmune panencephalitis and uveoretinitis transferred to the Lewis rat by T lymphocytes specific for the S100 beta molecule, a calcium binding protein of astroglia.

The pathogenic potential of autoimmune T cell responses to nonmyelin autoantigens was investigated in the Lewis rat using the astrocyte-derived calcium binding protein S100 beta, as a model nonmyelin autoantigen. The Lewis rat mounts a vigorous RT1B1 (major histocompatibility complex class II) restricted autoimmune response to an immunodominant S100 beta epitope (amino acid residues 76-91). The adoptive transfer of S100 beta-specific T cell lines induced a severe inflammatory response in the nervous system, but only minimal neurological dysfunction in naive syngeneic recipients. The inability of S100 beta-specific T cell transfer to induce severe disease was associated with a decreased recruitment of ED1+ macrophages into the central nervous system (CNS) in comparison with that seen in severe experimental autoimmune encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein (MBP)-specific T line cells. Moreover, unlike encephalitogenic MBP-specific T cell lines, S100 beta-specific T cell lines exhibited no cytotoxic activity in vitro. Histopathological analysis also revealed striking differences in the distribution of inflammatory lesions in MBP- and S100 beta-specific T cell-mediated disease. In contrast to the MBP paradigm, S100 beta-specific T cell transfer induces intense inflammation not only in the spinal cord, but throughout the entire CNS and also in the uvea and retina of the eye. In view of the distribution of lesions throughout the grey and white matter of the CNS we propose to term this new model experimental autoimmune panencephalomyelitis (EAP) to differentiate it from EAE. These experiments demonstrate for the first time that nonmyelin CNS autoantigens can initiate a pathogenic autoimmune T cell response, although the nature of the target autoantigen profoundly influences the clinical and histopathological characteristics of the resulting autoimmune disease. This is not simply a consequence of the distribution of the autoantigen, as both MBP and S100 beta are coexpressed in many areas of the CNS, but reflects differences in the capacity of different regions of the CNS to process and present specific autoantigens. This new model of T cell-mediated autoimmune CNS disease exhibits a number of similarities to multiple sclerosis (MS), such as its mild clinical course and the involvement of areas of the brain and eye, which are absent in myelin-mediated models of EAE. Nonmyelin autoantigens may therefore play an unexpectedly important role in the immunopathogenesis of inflammatory diseases of the CNS.

Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter.

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model.

Myotonic dystrophy (DM) is the most common form of muscular dystrophy and is caused by expansion of a CTG trinucleotide repeat on human chromosome 19. Patients with DM develop atrioventricular conduction disturbances, the principal cardiac manifestation of this disease. The etiology of the pathophysiological changes observed in DM has yet to be resolved. Haploinsufficiency of myotonic dystrophy protein kinase (DMPK), DM locus-associated homeodomain protein (DMAHP) and/or titration of RNA-binding proteins by expanded CUG sequences have been hypothesized to underlie the multi-system defects observed in DM. Using an in vivo murine electrophysiology study, we show that cardiac conduction is exquisitely sensitive to DMPK gene dosage. DMPK-/- mice develop cardiac conduction defects which include first-, second-, and third-degree atrioventricular (A-V) block. Our results demonstrate that the A-V node and the His-Purkinje regions of the conduction system are specifically compromised by DMPK loss. Importantly, DMPK+/- mice develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients. These results demonstrate that DMPK dosage is a critical element modulating cardiac conduction integrity and conclusively link haploinsufficiency of DMPK with cardiac disease in myotonic dystrophy.

The 1.1 A crystal structure of the neuronal acetylcholine receptor antagonist, alpha-conotoxin PnIA from Conus pennaceus.

BACKGROUND: alpha-Conotoxins are peptide toxins, isolated from Conus snails, that block the nicotinic acetylcholine receptor (nAChR). The 16-residue peptides PnIA and PnIB from Conus pennaceus incorporate the same disulfide framework as other alpha-conotoxins but differ in function from most alpha-conotoxins by blocking the neuronal nAChR, rather than the skeletal muscle subtype. The crystal structure determination of PnIA was undertaken to identify structural and surface features that might be important for biological activity. RESULTS: The 1.1 A crystal structure of synthetic PnIA was determined by direct methods using the Shake-and-Bake program. The three-dimensional structure incorporates a beta turn followed by two alpha-helical turns. The conformation is stabilised by two disulfide bridges that form the interior of the molecule, with all other side chains oriented outwards. CONCLUSIONS: The compact architecture of the PnIA toxin provides a rigid framework for presentation of chemical groups that are required for activity. The structure is characterized by distinct hydrophobic and polar surfaces; a 16 A separation of the sole positive and negative charges (these two charged residues being located at opposite ends of the molecule); a hydrophobic region and a protruding tyrosine side chain. These features may be important for the specific interaction of PnIA with neuronal nAChR.

Ventricular arrhythmia vulnerability in cardiomyopathic mice with homozygous mutant Myosin-binding protein C gene.

BACKGROUND: Homozygous mutant mice expressing a truncated form of myosin-binding protein C (MyBP-C(t/t)) develop severe dilated cardiomyopathy, whereas the heterozygous mutation (MyBP-C(t/+)) causes mild hypertrophic cardiomyopathy. Adult male MyBP-C(t/t) and MyBP-C(t/+) mice were evaluated for arrhythmia vulnerability with an in vivo electrophysiology study. METHODS AND RESULTS: Surface ECGs were obtained for heart rate, rhythm, and conduction intervals. Atrial, atrioventricular, and ventricular conduction parameters and refractoriness were assessed in 22 MyBP-C(t/t), 10 MyBP-C(t/+), and 17 wild-type MyBP-C(+/+) mice with endocardial pacing and intracardiac electrogram recording. Arrhythmia induction was attempted with standardized programmed stimulation at baseline and with isoproterenol. Heart rate variability and ambient arrhythmia activity were assessed with telemetric ECG monitors. Quantitative histological characterization was performed on serial sections of excised hearts. MyBP-C(t/t) and MyBP-C(t/+) mice have normal ECG intervals and sinus node, atrial, and ventricular conduction and refractoriness. Ventricular tachycardia was reproducibly inducible in 14 of 22 MyBP-C(t/t) mice (64%) during programmed stimulation, compared with 2 of 10 MyBP-C(t/+) mice (20%) and 0 of 17 wild-type controls (P<0.001). Ventricular ectopy was present only in MyBP-C(t/t) mice during ambulatory ECG recordings. There were no differences in heart rate variability parameters. Interstitial fibrosis correlated with genotype but did not predict arrhythmia susceptibility within the MyBP-C(t/t) group. CONCLUSIONS: MyBP-C(t/t) mice, despite prominent histopathology and ventricular dysfunction, exhibit normal conduction and refractoriness, yet are vulnerable to ventricular arrhythmias. Somatic influences between genetically identical mutant mice most likely account for variability in arrhythmia susceptibility. A sarcomeric protein gene mutation leads to a dilated cardiomyopathy and ventricular arrhythmia vulnerability phenotype.

Evaluation of the role of I(KACh) in atrial fibrillation using a mouse knockout model.

OBJECTIVES: We sought to study the role of I(KACh) in atrial fibrillation (AF) and the potential electrophysiologic effects of a specific I(KACh) antagonist. BACKGROUND: I(KACh) mediates much of the cardiac responses to vagal stimulation. Vagal stimulation predisposes to AF, but the specific role of I(KACh) in the generation of AF and the electrophysiologic effects of specific I(KACh) blockade have not been studied. METHODS: Adult wild-type (WT) and I(KACh)-deficient knockout (KO) mice were studied in the absence and presence of the muscarinic receptor agonist carbachol. The electrophysiologic features of KO mice were compared with those of WT mice to assess the potential effects of a specific I(KACh) antagonist. RESULTS: Atrial fibrillation lasting for a mean of 5.7+/-11 min was initiated in 10 of 14 WT mice in the presence of carbachol, but not in the absence of carbachol. Atrial arrhythmia could not be induced in KO mice. Ventricular tachyarrhythmia could not be induced in either type of mouse. Sinus node recovery times after carbachol and sinus cycle lengths were shorter and ventricular effective refractory periods were greater in KO mice than in WT mice. There was no significant difference between KO and WT mice in AV node function. CONCLUSIONS: Activation of I(KACh) predisposed to AF and lack of I(KACh) prevented AF. It is likely that I(KACh) plays a crucial role in the generation of AF in mice. Specific I(KACh) blockers might be useful for the treatment of AF without significant adverse effects on the atrioventricular node or the ventricles.

Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: a model for the role of disulfide bonds in structural stability.

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13)

Induction of atrial tachycardia and fibrillation in the mouse heart.

BACKGROUND: Atrial tachycardia and fibrillation in humans may be partly consequent to vagal stimulation. Induction of fibrillation in the small heart is considered to be impossible due to lack of a critical mass of > 100-200 mm2. Even with the recent progression of the technology of in vivo and in vitro mouse electrophysiological studies, few reports describe atrial tachycardia or fibrillation in mice. The purpose of this study was to attempt provocation of atrial tachyarrhythmia in mice using transvenous pacing following cholinergic stimulation. METHODS AND RESULTS: In vivo electrophysiology studies were performed in 14 normal mice. A six-lead ECG was recorded from surface limb leads, and an octapolar electrode catheter was inserted via jugular vein cutdown approach for simultaneous atrial and ventricular endocardial recording and pacing. Atrial tachycardia and fibrillation were inducible in one mouse at baseline electrophysiology study and eleven of fourteen mice after carbamyl choline injection. The mean duration of atrial tachycardia was 126 +/- 384 s. The longest episode lasted 35 min and only terminated after atropine injection. Reinduction of atrial tachycardia after administration of atropine was not possible. CONCLUSION: Despite the small mass of the normal mouse atria, sustained atrial tachycardia and fibrillation can be easily and reproducibly inducible with endocardial pacing after cholinergic agonist administration. This finding may contribute to our understanding of the classical theories of arrhythmogenesis and critical substrates necessary for sustaining microreentrant circuits. The techniques of transcatheter parasympathetic agonist-mediated atrial tachycardia induction may be valuable in further murine electrophysiological studies, especially mutant models with potential atrial arrhythmia phenotypes.

Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury.

Microglial proliferation and activation are common events in the injured CNS. The mechanisms, however, by which activated microglia are eliminated following a pathological stimulus are still poorly understood. The present study has therefore examined microglial proliferation by 3H-thymidine autoradiography and programmed cell death by terminal transferase-mediated nick end labeling (TUNEL) and in situ end labeling (ISEL) of nuclear DNA fragments in two models of peripheral nerve injury, i.e. sciatic and hypoglossal nerve transection in the rat. In these models, microglial activation and proliferation occur in CNS projection areas, i.e. in the ventral and dorsal gray matter of lumbar spinal cord and in the nucleus gracilis after sciatic nerve transection as well as in the axotomized hypoglossal nucleus. At these sites, microglial proliferation had a relatively sharp peak between days 2 and 3 post-lesion and then rapidly declined. DNA fragmentation was detected in lectin (GSI-B4)-positive microglia from day 6 after axotomy onward, reached an apparent peak at day 21 and was downregulated by day 60, i.e. the latest time point investigated. However, the expression of bcl-2 and c-myc, i.e. genes potentially controlling programmed cell death, was found to be unchanged during this period. Programmed cell death thus appears to be one mechanism by which activated microglia are gradually eliminated following CNS injury and steady state of microglial cell numbers is achieved in vivo. Expression of microglial growth factors may be instrumental in controlling these processes.

Ludwig Merzbacher (1875-1942): the man behind the disease.

Ludwig Merzbacher (1875-1942) is widely known for his seminal work on the pathology of the dysmyelinating CNS disease named for the clinician Friedrich Pelizaeus and himself. Yet his training, his scientific achievements and his list of publications suggest a scientist with broad interests in neuropathology, neuroscience, neurology and psychiatry. Among several studies in experimental and clinical neuropathology, Merzbacher's work on scavenger cells is the most outstanding. While working in Alois Alzheimer's laboratory in Munich in 1906/1907, Ludwig Merzbacher analyzed in great detail the reaction patterns of these cells, which are nowadays known as reactive microglia, and already attempted to elucidate their function in brain pathology.

Microglial reaction in the rat cerebral cortex induced by cortical spreading depression.

The response of microglial cells to cortical spreading depression (CSD) was studied in rat brain by immunocytochemistry. CSD was elicited for one hour by the topical application of 4M potassium chloride solution and the microglial reaction examined immunocytochemically after 4, 16, 24 and 72 hours. CSD was sufficient to induce a microglial reaction throughout the cortex at 24 hours. Activated microglial cells furthermore showed a striking de-novo expression of major histocompatibility complex class II antigens. In contrast, no microglial reaction was observed in the cortex of sham-operated animals. This microglial reaction in response to CSD was not associated with histologically detectable neuronal damage. These results support the view that microglial cells are extremely sensitive to changes of the brain microenvironment. Their activation may be related to changes of ion homeostasis in the brain which are not sufficient to trigger neuronal injury.

Astrocytes upregulate glial fibrillary acidic protein (GFAP), but not insulin-like growth factor-I (IGF-I) during experimental autoimmune neuritis (EAN).

T cell-mediated autoimmune neuritis produces rapid activation of spinal cord microglia. To determine whether this microglial response upregulates astrocytic expression of IGF-related proteins, we induced EAN and used in situ hybridization and immunocytochemistry to examine the mRNAs and peptides for glial fibrillary acidic protein (GFAP), insulin-like growth factor-I (IGF-I), IGF-I receptor (IGFR-I) and IGF binding protein-2 (IGFBP-2). Relative levels of GFAP mRNA and peptide were highest in the lumbar spinal cord 4-10 d following T cell transfer and significant GFAP elevations were still present after three weeks. The astrocytes expressing GFAP mRNA and peptide were localized around motoneurons which were related topographically to axons in peripheral nerve inflammatory lesions. In the nucleus gracilis, where terminals of dorsal root ganglion neurons are located, astrocytic levels of GFAP mRNA and peptide rose later and did not reach their highest levels until 21 d after T cell transfer. Even though microglia were activated in both locations 2-4 d after transfer, astrocytic levels of IGF-I, IGFR-I and IGFBP-2 mRNA and peptide did not differ significantly from those observed in controls. The dissociation of GFAP and IGF-I expression in EAN suggests that these astrocytic responses may be independently regulated. We also suggest that the type and severity of remote neuronal injury are probably more important inducers and regulators of these astrocytic responses than microglial cell activation.

Glial expression of the beta-amyloid precursor protein (APP) in global ischemia.

The beta-amyloid precursor protein (APP) bears characteristics of an acute-phase protein and therefore is likely to be involved in the glial response to brain injury. In the brain, APP is rapidly synthesized by activated glial cells in response to comparatively mild neuronal lesions, e.g., a remote peripheral nerve injury. Perfusion deficits in the brain result largely in neuronal necrosis and are a common condition in elderly patients. This neuronal necrosis is accompanied by a pronounced reaction of astrocytes and microglia, which can also be observed in animal models. We have therefore studied in the rat, immunocytochemically, the induction of APP after 30 min of global ischemia caused by four-vessel occlusion. The postischemic brain injuries were examined at survival times from 12 h to 7 days. From day 3 onward, APP immunoreactivity was strongly induced in the CA1 and CA4 regions of the rat dorsal hippocampus as well as in the dorsolateral striatum. In these areas, the majority of APP-immunoreactive cells were reactive glial fibrillary acidic protein (GFAP)-positive astrocytes, as shown by double-immunofluorescence labeling for GFAP and APP. Additionally, small ramified cells, most likely activated microglia, expressed APP immunoreactivity. In contrast, in the parietal cortex, APP immunoreactivity occurred focally in clusters of activated microglia rather than in astrocytes, as demonstrated by double-immunofluorescence labeling for APP and the microglia-binding lectin Griffonia simplicifolia isolectin B4. In conclusion, following global ischemia, APP is induced in reactive glial cells with spatial differences in the distribution pattern of APP induction in astrocytes and microglia.

Immunocytochemical study of an early microglial activation in ischemia.

Transient arrest of the cerebral blood circulation results in neuronal cell death in selectively vulnerable regions of the rat brain. To elucidate further the involvement of glial cells in this pathology, we have studied the temporal and spatial distribution pattern of activated microglial cells in several regions of the ischemic rat brain. Transient global ischemia was produced in rats by 30 min of a four-vessel occlusion. Survival times were 1, 3, and 7 days after the ischemic injury. The microglial reaction was studied immunocytochemically using several monoclonal antibodies, e.g., against CR3 complement receptor and major histocompatibility complex (MHC) antigens. Two recently produced monoclonal antibodies against rat microglial cells, designated MUC 101 and 102, were also used to identify microglial cells. Following ischemia, the microglial reaction was correlated with the development of neuronal damage. The earliest presence of activated microglial cells was observed in the dorsolateral striatum, the CA1 area, and the dentate hilus of the dorsal hippocampus. However, the microglial reaction was not confined to areas showing selective neuronal damage, but also occurred in regions that are rather resistant to ischemia, such as the CA3 area. Particularly in the frontoparietal cortex, the appearance of MHC class II-positive microglial cells provided an early indication of the subsequent distribution pattern of neuronal damage. The microglial reaction would thus seem to be an early, sensitive, and reliable marker for the occurrence of neuronal damage in ischemia.

Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline.

Ischemia-induced nerve cell death can partly be prevented by propentofylline, a pharmacon structurally related to xanthine derivates that interacts with the neuromodulatory function of endogenous adenosine. To evaluate a possible mechanism of neuroprotection by propentofylline, we studied its effect on the cellular production of reactive oxygen intermediates in microglial cells, which under pathological conditions can differentiate into brain macrophages, in comparison to peritoneal macrophages. Using a flow cytometric assay, we determined the intracellular formation of reactive oxygen intermediates by measuring the oxidation of the membrane-permeable and nonfluorescent dihydrorhodamine 123 to the cationic and intracellularly trapped, green fluorescent rhodamine 123 in single viable cells. Propentofylline at the therapeutic concentration of 50 microM completely inhibited the Ca(2+)-dependent Con A-induced increase in the production of reactive oxygen intermediates in peritoneal macrophages. In isolated and cultured microglial cells, which have a high spontaneous respiratory burst activity, the spontaneous production of reactive oxygen intermediates was reduced by approximately 30%. A phorbol 12-myristate 13-acetate-induced rise in the respiratory burst activity could not be inhibited by propentofylline in either cell type. An increased generation of reactive oxygen intermediates is thought to contribute to nerve cell death after brain ischemia, edema, and neurodegenerative diseases like Alzheimer's disease. These pathological conditions are all accompanied by an activation of microglial cells. We therefore suggest that the neuroprotective properties of propentofylline might in part be due to a modulation of the microglial production of potentially harmful reactive oxygen intermediates.

Transferrin Receptor Expression and Iron Uptake in the Injured and Regenerating Rat Sciatic Nerve.

Iron-saturated transferrin is a ubiquitous growth factor that plays a critical role in cellular iron uptake, growth and proliferation. Here we have studied the expression and distribution of transferrin receptors and iron uptake following injury of the rat sciatic nerve. Axotomy led to a massive but transient increase (days 2 - 9, maximum day 4) in [125I]transferrin binding at the site of the injury and in the distal, denervated part of the crushed or resected sciatic nerve, shortly preceding the time course of cellular proliferation (Friede and Johnstone, Acta Neuropathol, 7, 218 - 231, 1967; Jurecka et al., Acta Neuropathol, 32, 299 - 312, 1975). An additional, transient increase in specific binding was observed during reinnervation after reconnection of the resected sciatic nerve. Immunocytochemistry using the Ox-26 monoclonal antibody revealed strong and simultaneous expression of the transferrin receptor protein on two different cell types: on a subpopulation of blood-borne macrophages invading the injured peripheral nerve and on Schwann cells reacting to denervation and reinnervation. In addition, studies using intravenously injected radioactive iron (59Fe3+) showed a massive increase in endoneural iron uptake confined to the lesion site and to the distal part of the axotomised sciatic nerve, parallel to the time course of reactive transferrin receptor expression. Since iron is an essential cofactor of a number of key enzymes needed in energy metabolism and DNA synthesis, these data suggest that the induction of transferrin receptor expression may play an important role in the regulation of cellular growth and proliferation during peripheral nerve regeneration.

Characterisation of two new monoclonal antibodies directed against rat microglia.

With the aid of cultured rat microglial cells as immunogen, we raised two monoclonal antibodies, designated murine clone (MUC) 101 and 102, which recognised subsets of resident microglial cells in the normal central nervous system and cells of the mononuclear phagocyte system in peripheral organs. These antibodies were characterised by immunoperoxidase immunocytochemistry, immunoelectron microscopy, and immunoblotting. The immunostained cells were identified as microglial cells by double-immunofluorescence labelling with the B4-isolectin from Griffonia simplicifolia, an established microglial cell marker. Under normal conditions, both antibodies labeled resident microglia but with different distribution patterns. Under pathological conditions, e.g., after facial nerve transection, they labeled activated, perineuronal microglia in the operated facial nucleus. Immunoelectron microscopy demonstrated a membrane localisation of the antigen recognised by MUC 102. In peripheral organs, MUC 101 and 102 reacted with different cell populations of the mononuclear phagocyte system, particularly in thymus, spleen, and peripheral lymph node. Western blot experiments showed that MUC 101 recognised two proteins of 116 and 95 kD in fractions obtained from operated facial nucleus while MUC 102 reacted with two proteins of 62 and 70 kD molecular weight. These immunocytochemical results 1) confirm the antigenic similarity between microglia and cells of the monocyte-macrophage cell lineage, and 2) indicate that considerable antigen heterogeneity might exist among resident microglia. MUC 101 and 102 could thus become useful for studying the function of microglial cells both under normal and pathological conditions.

Solution structure of alpha-conotoxin ImI by 1H nuclear magnetic resonance.

alpha-Conotoxin ImI derives from the venom of Conus imperialis and is the first and only small-peptide ligand that selectively binds to the neuronal alpha7 homopentameric subtype of the nicotinic acetylcholine receptor (nAChR). This receptor subtype is a possible drug target for several neurological disorders. The cysteines are connected in the pairs Cys2-Cys8 and Cys3-Cys12. To date it is the only alpha-conotoxin with a 4/3 residue spacing between the cysteines. The structure of ImI has been determined by 1H NMR spectroscopy in aqueous solution. The NMR structure is of high quality, with a backbone pairwise rmsd of 0.34 A for a family of 19 structures, and comprises primarily a series of nested beta turns. Addition of organic solvent does not perturb the solution structure. The first eight residues of ImI are identical to the larger, but related, conotoxin EpI and adopt a similar structure, despite a truncated second loop. Residues important for binding of ImI to the alpha7 nAChR are all clustered on one face of the molecule. Once further binding data for EpI and ImI are available, the ImI structure will allow for design of novel alpha7 nAChR-specific agonists and antagonists with a wide range of potential pharmaceutical applications.