The synthesis of trans-[RuCl(NO)(cyclam)]2+ (cyclam = 1,4,8,11-tetraazacyclotetradecane) can be accomplished by either the addition of cyclam to K2[RuCl5NO] or by the addition of NO to trans-[RuCl(CF3SO3)(cyclam)](CF3-SO3). Crystals of trans-[RuCl(NO)(cyclam)](ClO4)2 form in the monoclinic space group P2(1)/c, with unit cell parameters of a = 7.66500(2) A, b = 24.7244(1) A, c = 16.2871(2) A, beta = 95.2550(10) degrees, and Z = 4. One of the two independent molecules in the unit cell lies disordered on a center of symmetry. For the ion in the general position, the Ru-N and N-O bond distances and the [Ru-N-O]3+ bond angle are 1.747(4) A, 1.128(5) A, 178.0(4) degrees, respectively. In both ions, cyclam adopts the (R,R,S,S) configuration, which is also consistent with 2D COSY 1H NMR studies in aqueous solution. Reduction (E degree = -0.1 V) results in the rapid loss of Cl- by first-order kinetics with k = 1.5 s-1 and the slower loss of NO (k = 6.10 x 10(-4) s-1, delta H++ = 15.3 kcal mol-1, delta S++ = -21.8 cal mol-1 K-1). The slow release of NO following reduction causes trans-[RuCl(NO)(cyclam)]2+ to be a promising controlled-release NO prodrug for vasodilation and other purposes. Unlike the related complex trans-[Ru(NO)(NH3)4(P(OEt)3)](PF6)2, trans-[RuCl(NO)(cyclam)]Cl2 is inactive in modulating evoked potentials recorded from mice hippocampal slices probably because of the slower dissociation of NO following reduction.

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Trans-spinal direct current (tsDC) stimulation is a modulator of spinal excitability and can influence cortically elicited muscle contraction in a polarity-dependent fashion. When combined with low-frequency repetitive cortical stimulation, cathodal tsDC [tsDC(-)] produces a long-term facilitation of cortically elicited muscle actions. We investigated the ability of this combined stimulation paradigm to facilitate cortically elicited muscle actions in spinal cord-injured and noninjured animals. The effect of tsDC-applied alone or in combination with repetitive spinal stimulation (rSS) on the release of the glutamate analog, D-2,3-(3)H-aspartate (D-Asp), from spinal cord preparations in vitro-was also tested. In noninjured animals, tsDC (-2 mA) reproducibly potentiated cortically elicited contractions of contralateral and ipsilateral muscles tested at various levels of baseline muscle contraction forces. Cortically elicited muscle responses in animals with contusive and hemisectioned spinal cord injuries (SCIs) were similarly potentiated. The combined paradigm of stimulation caused long-lasting potentiation of cortically elicited bilateral muscle contraction in injured and noninjured animals. Additional analysis suggests that at higher baseline forces, tsDC(-) application does not increase the rising slope of the muscle contraction but causes repeated firing of the same motor units. Both cathodal and anodal stimulations induced a significant increase of D-Asp release in vitro. The effect of the combined paradigm of stimulation (tsDC and rSS) on the concentration of extracellular D-Asp was polarity dependent. These results indicate that tsDC can powerfully modulate the responsiveness of spinal cord neurons. The results obtained from the in vitro preparation suggest that the changes in neuronal excitability were correlated with an increased concentration of extracellular glutamate. The combined paradigm of stimulation, used in our experiments, could be noninvasively applied to restore motor control in humans with SCI.

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The influence of melatonin on hippocampal evoked potentials initiated by low- and high-frequency electrical stimulations and by two pulses applied in rapid succession was investigated. In confirmation of our previous studies, melatonin attenuated the population spike triggered by low-frequency stimulation (0.03 Hz). High-frequency stimulation (HFS; 100 Hz for 1 sec, three times every 10 sec), which in control slices permanently facilitated neuronal excitability (347% +/- 32%), was also able to amplify the melatonin-depressed potential (467.8% +/- 59.6%). Because melatonin is a hydrophobic molecule, it was dissolved and applied in ethanol. Ethanol (0.4%) by itself reduced the magnitude of HFS-induced potentiation (233.5% +/- 16.8%). The slices stimulated with two pulses separated with a delay longer than 15 msec demonstrated a facilitation of the response to the second stimuli (paired-pulse facilitation; PPF). The influence of melatonin (100 microM) on PPF was biphasic: Shortly after addition of melatonin, PPF was briefly (5-10 min) reversed to paired-pulse inhibition (PPI), which gradually returned to a stable PPF. Ethanol (0.4%) applied without melatonin exerted only a marginal, facilitatory effect on PPF. The delay between two successively applied pulses, shorter than 13 msec, resulted in attenuation of the response to the second stimuli (PPI). Melatonin (100 microM) reversed the attenuation of the second potential within 15-20 min following its application. Ethanol applied by itself at the concentration of 0.4% temporarily (5-10 min), but significantly, depressed the second potential. These results demonstrate the ability of melatonin to modulate specific forms of plasticity in hippocampal pyramidal neurons.

The influence of melatonin on evoked potentials recorded from the CAI field of mouse hippocampal slices was investigated. Melatonin (0.1-2.0 mM) and its analog, 6-chloromelatonin (0.1-0.5 mM) depressed evoked potentials (EPSP and the population spike) in a concentration-dependent manner. The melatonin-induced depression was followed by a slow recovery phase. Since the fiber potential was not affected, it was concluded that melatonin influenced synaptic efficiency and/or cell excitability. Luzindole, an antagonist of MT2 melatonin receptors, although slightly depressing evoked potentials when applied by itself (100 microM), blocked any further inhibition by melatonin when added afterwards. We concluded that melatonin reduced synaptic efficiency and/or excitability of hippocampal neurons most likely through interaction with MT2 melatonin receptors, but other possible mechanisms of melatonin action are also considered.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.The long-term potentiation (LTP) of synaptic strength in hippocampal pyramidal neurons is a neurophysiological process iinplicated in the formation of memory traces in the brain (1, 2). While the initial chain of events that triggers this process is well-known, the mechanisms that determine the duration and stability of LTP have not yet been fully elucidated. Although it is generally accepted that the phosphorylation of proteins by several different kinases is involved in this stabilization, their exact localization and roles are still obscure (2, 3). Protein phosphorylation, a ubiquitous step in intracellular pathways that produce transient changes in neuronal activity, was found to serve also as a key mechanism of molecular adaptation in processes underlying the induction of longlasting alterations in synaptic function (for reviews, see ref. 4), including learning and memory formation (5-7). The first study that implicated protein phosphorylation in the process of learning (5) identified a synaptic phosphoprotein that was later shown to be a major neuronal substrate of protein kinase C (PKC), that plays a role in the maintenance-phase of LTP (8, 9). The specific PKC-isozyme involved in the maintenance of LTP was reported to be PKC C, a member of the atypical class (not stimulated by diacylglycerol or phorbol ...