Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells.

Disinhibition of cortical excitatory cell gate information flow through and between cortical columns. The major contribution of Martinotti cells (MC) is providing dendritic inhibition to excitatory neurons and therefore they are a main component of disinhibitory connections. Here we show by means of optogenetics that MC in layers II/III of the mouse primary somatosensory cortex are inhibited by both parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing cells. Paired recordings revealed stronger synaptic input onto MC from PV cells than from VIP cells. Moreover, PV cell input showed frequency-independent depression, whereas VIP cell input facilitated at high frequencies. These differences in the properties of the two unitary connections enable disinhibition with distinct temporal features.

Radiation-induced injury to the carotid artery: a longitudinal study.

Therapeutic radiation can lead to damage of arterial walls. In the present investigation, we studied prospectively 16 patients without pre-existing vascular disease (13 males, three females; mean age = 59.75 ± 11.9 years) who had undergone surgery and post-operative radiation therapy (cumulative dose = 56.2 ± 10.2 Gy) for the treatment of carcinoma of the head and neck. The carotid arteries were examined every 3 months for 19.9 ± 8.7 months using Doppler ultrasound and B-mode sonography. The thickness of the arterial walls was determined using an index (outer:inner mean arterial wall), and it was compared to a group of age-matched control subjects (n = 16) with healthy arteries. The index of the common carotid artery (CCA) was initially 1.14 ± 0.04 in both the right and left CCA in the radiation-treated subjects. By the end of the study, this index had increased significantly to 1.37 ˙ 0.14 in both right and left CCA in the radiation-treated subjects (p < 0.01). There was a significant difference in arterial wall thickness between the radiation-treated and control subjects (p < 0.001). Frequent and extensive smooth-surfaced plaques indicative of arterial thickening in the area of the common carotid and internal carotid arteries were seen in the irradiated patients. Four patients developed > 50% atherosclerotic stenosis of the internal carotid artery. These data suggest that arterial damage may occur following radiation treatment of the head and neck area. Modification of radiation therapy for the post-surgical treatment of head tumors coupled with the continuous monitoring of the carotid arteries using B-mode sonography could minimize this arterial damage.

Glutamic acid decarboxylase (GAD)-immunoreactive structures in the adult human lateral geniculate nucleus.

The distribution of glutamic acid decarboxylase (GAD)-like immunoreactivity in the lateral geniculate nucleus (LGN) of the human adult was studied in vibratome sections (50-60 microns thick) using the avidinbiotin-peroxidase method. The tissue was obtained at autopsy from five individuals without any known neurological disorders. Only few GAD-immunoreactive neurons were present in the layers of the LGN, even less in the interlaminar zones. The numerical density of GAD-immunoreactive neurons and puncta (probably synaptic boutons and or cross sectioned cell processes) in the magnocellular layers was larger than in the parvocellular layers. Furthermore, no striking differences between the individual parvocellular layers were noted. The immunoreactive somata were polygonal or triangular, occasionally pear-shaped, and ranged in size from 15 to 25 microns. They gave off two to four short, thick, straight primary dendrites. A preferred orientation of dendrites was not recognized. After bleaching the chromogen 4-chloro-1-naphthol and staining for lipofuscin pigment granules and basophilic material, 254 unequivocally relocated GAD-immunoreactive nerve cells could be classified as belonging to the lipofuscin pigment granules-containing class of interneurons.

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. : Characterization, Solubilization, and Photoaffinity Labeling.

Using the novel radioligand, [(3)H]-9'-nor-fusicoccin-8'-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K(+), Mg(2+)-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [(3)H]-9'-nor-fusicoccin-8'-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (K(D)), K(D1) = 1.5 nanomolar and K(D2) = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin.

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin, fusicoccin.

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60-70% of the complexes can be solubilized with 50-60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml(-1) soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9'-nor-fusicoccin-8'-alcohol-[(3)H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4-10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9'-nor-8'[(3,5-[(3)H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([(3)H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [(3)H]ABE-FC in the presence of 0.1-1 µM fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.

Characterization and localization of fusicoccin-binding sites in leaf tissues of Vicia faba L. probed with a novel radioligand.

Tritiated 9'-nor-fusicoccin-8'-alcohol provides a highly bioactive radioligand of high specific activity which is easily prepared by oxidation of fusicoccin and subsequent reduction with tritiated sodium borohydride. Using this radioligand, we have identified and characterized a selective binding site for fusicoccin (Ka for [(3)H]-9'-nor-fusicoccin-8'-alcohol=0.20·10(9) M(-1); Ka, apparent for fusicoccin=0.21·10(9) M(-1)) located at the plasmalemma of Vicia faba leaf tissue. The site is thermolabile, readily degraded by trypsin and located at the apoplastic face of the plasmalemma based on results obtained using right-side-out plasmalemma vesicles prepared by aqueous two-phase partitioning and macromolecular fusicoccin-derivatives. The binding-protein is present in guard cells of Vicia faba, as shown by the use of purified guard-cell protoplasts.

Monoclonal antibodies against fusicoccin with binding characteristics similar to the putative fusicoccin receptor of higher plants.

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [(3)H]-nor-fusicoccin-alcohol of high specific activity (1.5 x 10(14)Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 x 10(-9) molar and 1.85 x 10(-9) molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 x 10(-9) molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [(3)H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe.