Heterogeneity in KCL induced calcium fluctuations in cultured hippocampal cells

S36 nulated for blood pressure monitoring and ICP was elevated via the cisterna magna infusion method, up to 50–60 mm Hg for a period of 13 min. Blood flow decreased significantly , from 100%, down to 9.54 ± 3.6%, NADH increased significantly by 77 ± 11%. The extracellular levels of K+ and Ca2+ reached concentrations of 39.9 ± 6.7 and 0.013 ± 0.008 mM, respectively, and the pH decreased to 6.97 ± 0.076. During ICP elevation mean blood pressure stayed at a level of 100 ± 5.6 mm Hg. Recovery of all parameters was complete 10 min after the reopening of the cannula to the cisterna magna to air pressure. These findings suggest that intracranial hypertension for a period of 13 min causes reversible damage to the brain tissue. Supported by the Chief Scientist’s Office, Ministry of Health, and by the Research Authority, Bar-Ilan University, Ramat Gan, Israel. REACTION OF HUMAN BUTYRYLCHOLINESTERASE HIS117 ENZYMES WITH CARBAMATES C.B. Millard1,2, O. Lockridge3, I. Silman1 and C.A. Broomfield2 1 Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel; 2 U.S. Army Medical Research Institute of Chemical Defense, APG, MD, USA; 3 University. of Nebraska Medical Center, Institute of Eppley Cancer, Omaha, NE, USA By replacing a Gly in the ‘oxyanion hole’ of butyrylcholinesterase (BChE) with His, we designed two mutant enzymes (G117H and G117H/E197Q) that hydrolyze organophosphorus nerve agents and related ‘phosphotriester’ covalent inhibitors. This hydrolytic activity was not observed with wildtype (WT) BChE, G117K, or E197Q (Biochemistry 34: 15925–15933, 1995; 36: 786–795, 1997). Carbamates (CBs) also inhibit BChE via a covalent intermediate and are used as a protective pre-treatment for nerve agent poisoning. We report here on the reaction of the His-117 enzymes with two CBs, using butyrylthiocholine (BTCh) as substrate at pH 7.5, 25°C: physostigmine pyridostigmine ki k3 (m−1 min−1) (min−1) WT BChE 1050000 G117H 13600 G117K 14800 G117H/E197Q 14

0.0060 0.0018 0.0070 0.0005

ki k3 (M−1 min−1) (min−1) 10000 250 200 60

0.0024 0.0009 ND 0.0008

Carbamylation of G117H or G117K, as judged by ki, was much slower than that of WT. This may result from steric hindrance (physostigmine) or charge repulsion (pyridostygmine) in the reversible complex. In contrast, decarbamylation (k3) decreased only 2- to 3-fold for G117H and was almost unaffected for G117K, compared to WT. This was consistent with the 2.5-fold reduction in the kcat for BTCh reported for G117H. We conclude that oxyanion stabilization of the rate-limiting deacylation (BTCh) or decarbamylation transition states, both of which should be tetrahedral, is not greatly impeded by the additional His or Lys side chains. AUTOMATED DETECTION OF MULTIPLE SCLEROSIS LESIONS IN THE HUMAN BRAIN USING MR IMAGE PROCESSING S. Miron, D. Goldberg-Zimring§, H. Azhari§, A. Achiron Neuroimmunology Unit, Sheba Medical Center, Tel Hashomer, Israel; Dept. of Biomedical Engineering§, Technion, Haifa, Israel Multiple Sclerosis (MS) is a chronic demyelinating disease affecting the white matter of the central nervous system (CNS). Brain MRI is used for MS diagnosis, since the images provide a good contrast between MS lesions and the CNS white matter. The MS lesion usually appears on MRI as an area that during disease course can increase, decrease, or remain unmodified. A key issue in clinical studies evaluating disease progression by MRI is the quantification (area, number)

of MS lesions. Thus, a need exists for the development of accurate, reproducible, and quantitative techniques. In this study an automatic algorithm for detection and contouring of multiple sclerosis lesions is described. The algorithm was developed with the software Matlab (Version 5.0). The process consists of four main stages: (1) detection and contouring of all hyper-intense signal regions within the image; (2) elimination of false positive segments by size, and anatomical location; (3) removal of artifacts using an artificial neural paradigm (BackPropagation); (4) the operator can remove artifacts or add undetected lesions. The algorithm was applied to 48 MR images obtained from 12 patients with relapsing remitting MS. Images were acquired on a high field MRI scanner (2T Prestige, Elscint). The algorithm successfully detected 92 ± 15.9% of the lesions and eliminated 93 ± 4.6% of the artifacts. Automatic detection of MRI lesions in MS improve accuracy and reproducibility compared to manual tracing methods. Hence, applying the algorithm in computerized MR image analysis may ameliorate the monitoring of disease burden in MS. POSTNATAL ENHANCEMENT OF HOST ACETYLCHOLINESTERASE IN DEVELOPING ACHE TRANSGENIC MICE I. Mor, S. Seidman and H. Soreq The Dept. of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel FVB/N mice overexpressing human (h) transgenic acetylcholinesterase (AChE; EC 3.1.1.7) in brain neurons develop normally but display late-onset impairments in cognitive and neuromotor performance. Prenatal enhancement in levels of mRNA encoding the acetylcholine synthesizing enzyme - choline acetyltransferase (ChAT), was interpreted as a compensation mechanism for sustaining cholinergic balance prior to birth. Here, we report our search for mechanisms enabling postnatal brain development under the hypocholinergic function caused by elevated levels of AChE. Transgenic (h)AChEmRNA levels, quantified by kinetic follow-up of RT-PCR amplification, remained constant throughout the 15 days after birth, at a time when murine (m)AChEmRNA levels increase by 10 fold. This suggested that the short 586 bp ACHE promoter directing expression of the human transgene did not enable developmentally regulated transcriptional control in the murine milieu. However, despite the excess AChEmRNA contributed by the transgene, brain AChE activity in the transgenic mice remained similar to that of controls until postnatal day 5, after which it abruptly increased to reach a persistent 2-fold excess by day 10, as compared to controls. In contrast, brain AChE levels in non-transgenic control mice increased throughout this period at a constant linear rate. This analysis demonstrates that the abrupt increase in AChE activity observed in the brains of transgenic mice was due to enhanced synthesis of murine AChE. We conclude that after postnatal day 5, excess host and transgenic enzyme production together create a cholinergic imbalance that ultimately leads to the late-onset neurodeterioration phenotype characteristic of these mice. HETEROGENEITY IN KCL INDUCED CALCIUM FLUCTUATIONS IN CULTURED HIPPOCAMPAL CELLS Y. Nazarian, S.G. Kachalsky and R. Rahamimoff Dept. of Physiology and The Bernard Katz Minerva Centre for Cell Biophysics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel We examined the effect of elevation in extracellular potassium concentration ([K+]out) on the intracellular calcium levels and fluctuations, using the calcium indicator Fluo-3 and confocal laser scanning microscopy (CLSM). Most hippocampal cells in culture respond to [K+]out elevation by increasing their calcium signals. In a minority of the cells, [K+]out elevation caused first a decrease in free intracellular calcium concentration ([Ca2+]in), followed by a slow increase. The oscillations in [Ca2+] in were altered in elevated [K+]out and showed a faster perio-

S37 dicity. Time series stochastic analysis was used to determine the ‘cross talk’ among different cells in culture. In a number of cases, elevation of [K+]out converted cells with negative cross correlation coefficient in the calcium signal to cells with a significant positive cross-correlation coefficient. These results indicate that elevation in [K+]out causes a calcium synchronization. PHARMACOLOGICAL STUDIES ON IONOTROPIC GLUTAMATE RECEPTORS. M. Neuman, M. Dahan-Fumbar and Y. Stern-Bach Dept. of Anatomy and Cell Biology, The Hebrew University - Hadassah Medical School, Jerusalem, Israel Ionotropic glutamate receptors are the primary mediators of excitatory synaptic transmission in the mammalian central nervous system (CNS). They also appear to play key roles in brain development, acquisition of memory, and in a number of chronic and acute neurological disorders. Glutamate receptor agonists and antagonists are thus of major interest both as experimental tools for investigating fundamental CNS function, and as potential therapeutic drugs for neuronal disorders. Several such compounds have been characterized over the years and allowed the separation of glutamate receptors into three pharmacological groups: AMPA, kainate and NMDA receptors. However, despite extensive characterization little is known about the interaction of these drugs with the receptor-protein itself. Using a series of chimeric proteins generated between two members of the family (GluR3, an AMPA receptor and GluR6, a kainate receptor) we identified two discontinuous segments of the glutamate receptor protein, which determine agonist specificity. These segments share structural similarities with a set of bacterial proteins involved in binding of various amino acids. Based upon the crystal structure available for one of them, we proposed a structural model for the glutamate binding site of the mammalian receptors (Stern-Bach et al., 1994, Neuron 13: 1345–1357). We are now using these functional chimeric proteins combined with the structural model, to study several novel subtype selective antagonists of AMPA and kainate receptors. In particular, we identify four discontinuous regions on the GluR3 subunit, which contribute to the inhibitory action of GYKI-53655, a non-competitive AMPA receptor antagonist.

VISUAL AND AUDITORY PROPERTIES OF CORTICAL CELLS IN THE SUPRASYLVIAN AREA OF THE CAT N. Notkin, R. Yaka and U. Yinon Physiol. Lab., Goldschleger Eye Research Institute, Tel-Aviv University Faculty of Medicine, Sheba Medical Center, Tel-Hashomer, Israel We have studied visual and auditory activity in the visual areas of the Lateral Suprasylvian Sulcus (PMLS- postero-medial, PLLS- postero-lateral, AMLS- antero-medial and ALLS- antero-lateral), in order to reveal possible multimodality there. Single cells were extracellularly recorded in the cortex of 14 adult cats (age: 1.5–2 years) following anesthesia and paralysis. Visual stimulation consisted of moving bars and acoustic stimuli were pure tones and clicks. Post-stimulus time histograms (PSTHs) of all responding cells were computed and analyzed. Visual areas

ALLS AMLS PLLS PMLS

Total number of cells

Responsiveness (%)

% of total responding cells Visual

Auditory

Bimodal

361 221 281 168

42.1 50.7 38.0 42.9

15.8 42.8 43.9 69.4

79.6 39.3 48.6 11.1

4.6 17.9 7.5 19.5

The results show that (see table): (1) All the above mentioned areas could be activated either uni- or bimodally by auditory and/or visual stimuli; (2) The multimodal responses were maximal in the medial areas (PMLS and AMLS); (3) The percentage of auditory cells was remarkably high in the ALLS (topographically related to the auditory cortex) and lowest in the PMLS; (4) The responsiveness in all areas was considerably low. In is thus evident that all four areas in the medial and lateral banks of the Lateral Suprasylvian Sulcus are polysensory. The low responsiveness obtained indicates that the somatosensory modality may be also involved. We acknowledge the assistance of Prof. Z. Wollberg in this study.

DIFFERENTIAL REGULATION OF ADENYLYL CYCLASE ISOZYMES UPON CHRONIC ACTIVATION OF INHIBITORY G PROTEIN-COUPLED RECEPTORS I. Nevo, T. Avidor-Reiss, R. Levy, M. Bayewitch and Z. Vogel Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

DIFFERENTIAL COUPLING OF D2-TYPE DOPAMINE RECEPTORS TO THE PTX-INSENSITIVE GZ GTP BINDING PROTEIN J. Obadiah, B. Levavi-Sivan, T. Avidor-Reiss1, M. Bayewitch1, Z. Vogel1, C.S. Fishburn and S. Fuchs Depts. of Immunology and Neurobiology1, Weizmann Institute of Science, Rehovot, Israel

While acute activation of opiate or other inhibitory Gi/o-coupled receptors generally leads to inhibition of adenylyl cyclase (AC) activity, chronic receptor activation was shown to lead to a time- and dosedependent activation of AC. This phenomenon, particularly manifest following agonist withdrawal, has been referred to as AC superactivation and is believed to play an important role in the development of opiate drug addiction and withdrawal. However, the mechanism by which this superactivation is evoked and the nature of the AC isozymes participating in the process remain largely unknown. Using COS-7 cells transfected with cDNAs of the various AC isozymes and the D2dopaminergic or m4-muscarinic receptors, we have shown that acute activation of these receptors leads to inhibition of the activity of AC isozymes I, V, VI and VIII, while types II, IV and VII are stimulated and type III is not affected. Conversely, chronic receptor activation leads to superactivation of AC types I, V, VI and VIII and to a reduction in the activities of types II, IV, and VII. No change was observed in AC-III activity. This pattern is similar to that recently observed by us upon acute and chronic activation of the µ-opiate receptor, suggesting that AC superactivation may represent a general means of cellular adaptation to chronic activation of inhibitory receptors, and that the AC response in different brain areas (or cell types) could be explained by the presence of different populations of AC isozymes in these areas.

The D2-type dopamine receptors are thought to inhibit adenylate cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. Gz is a member of the Gi family, expressed primarily in brain and platelets, and is insensitive to PTX. We were interested to find out whether and to what extent the various members of the D2 receptor family (D2S, D2L, D3S, D3L, or D4) can couple to Gz to produce inhibition of AC activity. COS 7 cells were transfected with cDNA of each of the above mentioned murine dopamine receptors alone, or together with the cDNA of the a subunit of Gz. The transfected cells were treated with PTX to inactivate endogenous Ai. Coupling of each of the receptor subtypes to Gi and to Gz was estimated by measuring the inhibition of cAMP accumulation induced by the dopaminergic agonist quinpirole (100 nM) in forskolin (FSK; 20 mM)stimulated cells. The murine D2S and D2L receptor isoforms which couple to Gi were shown to couple to Gz as well, as also reported previously for the rat D2L receptor. Similar levels of inhibition of AC were obtained in cells transfected with either D2S or D2L alone, or cotransfected with Gz, indicating that these receptors can couple equally to Gi or Gz. The murine D3S and D3L receptors did not couple to Gi in transfected cells, as the addition of quinpirole did not result in any significant inhibition of AC activity. Moreover, in the presence of PTX there