The neuroprotective effect of activity dependent neurotrophic protein in mice after closed head injury

S9 Total denervation of a limb or its part elicits in rodents a behavior of self mutilation of the denervated limb (autotomy). Many lines of evidence support the suggestion that this behavior is an expression of unagreeable dysesthetic sensations typical of neuropathic pain. Since catecholamines (CA) serve as antinociceptive agents in the CNS, and CA-reuptake blockers are effective analgesics in humans suffering from neuropathic pain and in related animal models, in the present study we examined the autotomy behavior in a rat line which has geneticallydetermined increased levels of CA in the CNS (Flinders Sensitive Line (FSL); n = 11). Autotomy was compared to a control group of the unselected progenitor Sprague–Dawley (SD) strain (n = 10). These rats underwent a total hindpaw denervation by sciatic and saphenous neurectomy. Autotomy behavior was followed three times a week, for 70 days postoperatively (PO), by two ‘blinded’ observers. Scoring of autotomy levels was done by assigning 1 point for mutilation of ≥2 nails and an additional point for each self-injured ½ toe, up to a cumulative maximum score of 11. In support of our hypothesis, we found that FSL rats expressed lower levels of autotomy compared to SD rats. The incidence of high autotomy levels (scores ≥ 6) in FSL rats, on day 70 PO, was 36% compared to 70% in SD rats (P = 0.01, χ2 test). The average day when high autotomy levels were first observed in FSL rats was 63.2 ± 3.3 compared to 44.8 ± 3.2 in SD rats (P < 0.05, t-test). We conclude that genetically-determined hypercatecholaminergia in the CNS provides animals with endogenous partial protection of neuropathic pain. Thus, FSL rats can be used as a line of choice for the study of the role of central catecholaminergic mechanisms in neuropathic pain. SYNERGISTIC ACTIONS OF GABA and GLUTAMATE, GIANT DEPOLARISING POTENTIALS & CA2+ OSCILLATIONS IN THE DEVELOPING HIPPOCAMPUS Y. Ben-Ari INSERM Unit 29, 123 Bd de Port-Royal , Paris, France GABA, the principal inhibitory transmitter in adult central nervous system, acts as an excitatory transmitter in early postnatal life 1. Recent studies have confirmed and extended this concept by showing that in the neonatal hippocampus, the main ionotropic channels (GABAA, NMDA and AMPA receptors) display a sequential participation to neuronal excitation; GABAA receptors facilitating the activation of NMDA receptors at a time when transmission via AMPA receptors is poorly developed. Furthermore, the neonatal circuit is characterised by the presence of network driven Giant Depolarising Potentials (GDPs) that are generated by the synergistic actions of GABA and NMDA receptors and associated with large Ca2+ oscillations. GDPs have now been reported in a wide range of developing structures and appear to be a basic feature of the developing brain that probably participates in the activity dependent formation of functional units. We have studied the generation and propagation of GDPs in the newly developed Intact Hippocampal Formation (IHF) that can be kept in vitro for long durations and the entire repertoire of techniques that have made slices so popular used (Khalilov et al., 1997); GDPs propagate from one hippocampus to the contralateral one and also to other limbic structures. The mechanisms underlying the development of this ‘menage à trois’ and the role of GDPs will be discussed. Ben-Ari et al., Trends Neurosci., in press, 1997. Khalilov et al., Neuron, in press, 1997. DECREASED ACTIVITY AND INCREASED SUSCEPTIBILITY TO DOPAMINE OF COMPLEX I IN SCHIZOPHRENIC PATIENTS D. Ben-Shachar, H. Gazawi, R. Zuk, A. Reshef, A. Sheinkman1, E. Klein Laboratory of Psychobiology, Dept. of Psychiatry, Bruce Rappaport Faculty of Medicine Technion, Rambam Medical Center, 1 Mental Health Center Tirat Carmel, Haifa, Israel

Impairment of mitochondria has been suggested to be involved in the pathogenesis of neurodegenerative diseases. In spite of numerous research efforts, no organic brain defects has been clearly correlated with schizophrenia. Thus, abnormal energy metabolism could provide an reasonable explanation for altered turnover of dopamine in schizophrenia. Recently, we have reported that dopamine had a profound inhibitory effect on mitochondrial NADH dehydrogenase (complex I) activity (IC50 = 8–10 PM) in vitro. Others have shown that high doses of dopamine inhibit complex I activity in vivo as well. Alterations in dopamine transmission are among the most consistent findings related to schizophrenia. Therefore, we have studied complex I activity, and its susceptibility to dopamine, in platelets isolated from schizophrenic patients. Complex I activity was increased (437 ± 22 vs. 191 ± 16 nmol/mg protein/min) and was more sensitive to dopamine inhibition as compared to age and sex matched controls (65.2 ± 5.1 vs. 38.6 ± 4.0%). Antipsychotic medication had no effect and drug free patients (for 1 month up to 15 years) showed a similar enhanced activity of complex I and the same susceptibility to dopamine as medicated patients. The alteration in complex I activity was specific, since complex I activity of patients with major depression or bipolar disorder was similar to that of controls. In addition, no change was observed in the activity of cytochrome c between the different patient groups and healthy controls. The results of this study suggest that cellular respiration is altered in schizophrenia and thus might be an important factor in the pathophysiology of the disorder. STIMULUS AND TASK INFLUENCES IN COVERT ORIENTING OF ATTENTION G. Ben-Yehudah and E. Zohary Dept. of Neurobiology, The Hebrew University of Jerusalem, Jerusalem, Israel Studies of attention consistently found that a peripheral visual cue facilitates rapid discrimination of a target in a cued location. We previously reported that in cases of spatial uncertainty, covert orienting of attention lead to gains in human psychophysical performance in a motion discrimination task. In addition to this validity effect, we observed a right visual field advantage in motion perception. We suggested that this visual field asymmetry might result from an inherent bias in the allocation of attention. To test the robustness of these effects, we examined them in two different conditions. First, we changed the focus of the task from discriminating the direction of motion to locating the relevant area of motion. In the motion discrimination task, a cue informed the subjects of the future location of the signal dots, and the subjects reported their direction of motion. In contrast, the motion localization task required the subjects to report the location of the signal dots, while the cue informed them of the signal dots future direction of motion. This change in task requirements abolished both the validity effect and the visual field asymmetry. In the second condition we changed the task stimulus, switching from motion discrimination to form discrimination. Although we altered the type of stimulus, from a moving random dot array to static tilted bar array, we still found a similar visual field asymmetry. Therefore, we suggest that discrimination tasks, which require attention, show both the validity effect and the visual field asymmetry. However, localization tasks, which do not require attention, show neither effect. THE NEUROPROTECTIVE EFFECT OF ACTIVITY DEPENDENT NEUROTROPHIC PROTEIN IN MICE AFTER CLOSED HEAD INJURY L. Beni-Adani1, S. Pomeranz1, I. Gozes2, M. Bassan2, D. E. Brenneman3 and E. Shohami4 Depts. of 1 Neurosurgery and 4 Pharmacology, The Hebrew University Hadassah Medical Center, Jerusalem, Israel; 2 Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 3 Section on Developmental and Molecular Pharmacology, LDN, NICHD, NIH, Bethesda, MD, USA

S10 Traumatic injury to the brain causes primary death of neural tissue, and secondary necrosis secondary due to locally-induced mechanisms. Vasoactive intestinal peptide was shown to be neuroprotective through the induced release of a family of activity-dependent-neurotrophicfactors from astroglial cells (JCI 97, 2299, 1996). In this family, activity dependent neuroprotective protein (ADNP) and peptide derivatives were shown to be effective in several pathologies (submitted). The role of ADNP as a neuroprotective agent in a mouse model of closed head injury (CHI) was explored. Sabra male mice were randomized for placebo and ADNP treatments 1–2 h after CHI. Their clinical status was determined by a Neurological Severity Score (NSS, scoring 1–10) at 1 h and at various intervals after CHI (24 h-30 days) and recovery was evaluated as the difference between NSS at 1 h and at any later time. Brain edema was determined as percent water content 24 h after CHI. Brains were fixed and stained with H&E at injured site of the cortex and the hippocampus, and percent dead neurons was calculated. For comparison, mice were matched by severity of trauma (NSS at 1 h) and by macroscopically and microscopically observations of the brains. Results: Mice treated with ADNP had a lower NSS (P < 0.05) and better recovery (P < 0.005) than controls. Water content (78.5 ± 0.3% in normal brain) was elevated after CHI to 84.43 ± 0.6% in vehicle-treated mice, and only to 80.23 ± 0.03% in ADNP-treated mice (P < 0.01). Hippocampal neuronal mortality was unaffected by the treatment in the severely traumatized mice, whereas a trend towards protection by ADNP was found after less severe injury. Conclusion: ADNP seems to be a promising neuroprotectant in mouse CHI model, resulting in improved neurological performance and dramatic reduction of edema. TASTE EXPERIENCE ACTIVATES MAP KINASE IN THE RAT INSULAR CORTEX D.E. Berman, S. Hazvi, R. Lamprecht, R. Seger and Y. Dudai Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

P.Z. Beserman, R. Hershkovitz, O. Lider, A. Kesler, S. Muller, D. L. Hirschberg and M. Schwartz* Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel In spite of being highly enriched in resident macrophages (microglia), the brain is a site that allows an extended survival time of immunogens (an immunologically privileged site). This has been suggested to be due to local immunosuppression of macrophage recruitment and activation. Here we report the discovery, in the mammalian central nervous system (CNS) but not in the peripheral nervous system (PNS), of a peptide that inhibits macrophage migration, activation as phagocytic cells, and adhesion to extracellular matrix (ECM) of both macrophages and T cells. The peptide was purified from adult rat optic nerves by size-exclusion chromatography, reversed-phase high-pressure liquid chromatography, and then layer chromatography. The presence of this inhibitor in the CNS may explain both the poor ability of bloodborne macrophages to invade the CNS and the permanent suppression of brain-resident microglia. It underlines the immune privilege of the CNS and was therefore named as immune privilege factor (IPF). It is further suggested that this peptide may be a biochemical link CNS immune privilege and the loss of ability of the mammalian CNS to regenerate. A decrease in its level may explain inflammation-associated disorders. EARLY APPEARANCE OF SPONTANEOUS SYNAPTIC ACTIVITY IN LAYER IV NEURONS OF DEVELOPING MOUSE BARREL CORTEX Y. Bibi, I.A. Fleidervish and M.J. Gutnick Zlotowski Center for Neuroscience and Dept. of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel

When rats sample an unfamiliar taste, protein phosphorylation and protein synthesis are modulated in the insular cortex, which contains the gustatory cortex (Rosenblum et al., Behav. Neural Biol. 59: 49–56, 1993; J. Neurosci. 17: 5129–5135, 1997). These changes probably subserve consolidation of short-term taste memory into a long-term form. Consolidation is expected to involve synaptic remodeling. Mitogen activated protein kinases (MAPKs) are intracellular kinases involved in cellular remodeling and differentiation in various tissues in response to growth factors, mitogens and neurotransmitters. We set out to investigate whether taste learning activates MAPK in the insular cortex. Rats were presented for 10 min with 10 ml of 0.1% saccharin, an unfamiliar taste. From previous experiments we know that such an exposure (i.e., incidental learning) suffices to form a robust memory of the novel taste. The rats were sacrificed at various times afterwards, and the insular cortex excised, homogenized, and immunoblotted with monoclonal antibodies recognizing the biphosphorylated, activated form of ERK1 and 2, members of the MAPK family. An increase in activated MAPK was observe at 10 min, reaching a maximum of 3.0 ± 0.7-fold at 30 min, and subsiding to basal levels within an hour. The total level of MAPK was not affected (1.1 ± 0.2). The effect was not observed in the cerebellum, occipital lobe, piriform cortex or olfactory bulbs. In order to visualize the activation in cortex, we performed immunocytochemical analysis of brain sections. The percentage of MAPK activated cells in the insular cortex 30 min after the first exposure to the novel taste increased from 23 ± 4% to 97 ± 5% (water vs. saccharin exposed animals, respectively). Patches of activated MAPK cells were observed in cortical layers III–V. Supported by the Green Foundation and the Reich Foundation, and by the Dominic Center for Higher Brain Function, Weizmann Institute of Science.

Layer IV neurons in mouse somatosensory cortex are arrayed in cell clusters known as barrels, which correspond topographically to the mystacial whiskers on the face. In a recent electron microscopic developmental study of layer IV barrel field, White et al. (Somatosens. Motor Res., 14: 34–55, 1997) reported a low density of excitatory (asymmetrical) and virtually no inhibitory (symmetrical) synapses on postnatal day 6 (P6), and a sharp rise during the second week of life. Here, we have sought to determine the physiological correlates of these changes. Spontaneous PSCs were monitored by whole cell patch clamp recording from layer IV neurons in 400 Pm tangential slices of barrel cortex from neonatal (P5–P6) and juvenile (P13–P15) mice. K+ currents were blocked with intracellular Cs+ and Na+ currents with QX-314. EPSCs were revealed by clamping the cells at −70 mV (E(GABA-A)) and IPSCs were demonstrated by clamping the cells at 0 mV (E(AMPA) and E(NMDA)). To study NMDA mediated EPSCs 10 PM BMI and 40 PM CNQX were added and events were recorded at +40 mV. Both sIPSCs and sEPSCs were already evident on P5. IPSC frequencies were between 0.26 and 26 Hz, EPSC frequencies were between 0.38 and 23 Hz. IPSC amplitudes were between 13 and 48.9 pA and EPSC amplitudes between −7.7 and −15.5 pA. The IPSC decay τ was 10–14 ms in both neonates and juveniles. The decay τ of NMDA EPSCs in neonates was > 200 ms at +40 mV, but < 50 ms in juveniles. Neither amplitudes nor frequencies of sPSCs changed significantly with age. These data show that excitatory and inhibitory synaptic activity is already prominent on P5, and that it does not appear to increase in parallel with the sharp morphological increase in numbers of synapses. The kinetics of the NMDA-mediated EPSCs, however, does undergo a maturational change, which presumably reflects a developmental molecular change in receptor composition. Supported by the Basic Research Fund of the Israel Academy of Sciences.

ISOLATION OF AN ABUNDANT CNS RESIDENT IMMUNOSUPPRESSIVE PEPTIDE: A BIOCHEMICAL BASIS FOR IMMUNE PRIVILEGE

CHARACTERISTICS OF SPONTANEOUS EXCITATORY SYNAPTIC CURRENTS IN LAYER IV NEOCORTICAL NEURONS