The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-I-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CAl and mossy fiber-CA3 pathways. t-PA-'-mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CAl and CA3 does not result in hippocampus-dependent behavioral phenotypes.The hippocampus of the mammalian brain contains three major neural pathways that undergo an activity-dependent change in synaptic strength, called long-term potentiation (LTP). In each of these pathways, hippocampal LTP consists of two components, an early, transient, transcriptionally-independent component [earlyphase LTP (E-LTP)] and a later, persistent, long-term component [late-phase LTP (L-LTP)] that requires protein and mRNA synthesis (1-3). Hippocampus-dependent long-term memory (4, 5), unlike short-term memory, also requires protein and mRNA synthesis, which implies that long-term memory requires the expression of specific genes and proteins (6, 7). The gene transcription required for long-term memory is thought to result from a leaming-induced increase in neuronal activity, which ultimately leads to the strengthening of preexisting synapses as well as MATERIALS AND METHODSElectrophysiology. Transverse hippocampal slices (400-500 ,tM) were placed in an interface chamber and perfused continuously with solution containing 124 mM NaCl, 1.3 mM MgSO4, 4 mM KCl, 1.0 mM Na2HPO4, 2.0 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose. The perfusion solution was bubbled with 95% 02 and 5% Co2 at a flow rate of 1.5-2 ml/min. The temperature of -the recording chamber was maintained at either 28°C or 32°C in different experiments. A stainless steel electrode was used for field excitatory postsynaptic potential (EPSP) recording, and a fine tungsten bipolar electrode was used for stimulation. The position of the electrodes for stimulating and recording in CAl and CA3 was the same as described (2,14). Test stimuli were of 0.05-ms pulse duration and 0.016-Hz frequency. LTP was induced by one or four trains of tetanus at the same intensity as test stimuli (100 Hz for 1 s, pulse duration 0.05 ms) in the Schaffer collateralCAl pathway and four trains of tetanus (100 Hz for 1 s) with double-pulse duration (0.1 ms) during tetanus in the mossy fiber-CA3 pathway. The initial slope of the EPSP was measured, and LTP was calculated as per...

To gain more insight in the physiological function of the fragile X gene (FMR1) and the mechanisms leading to fragile X syndrome, the Fmr1 gene has been inactivated in mice by gene targeting techniques. In the Morris water maze test, the Fmr1 knockout mice learn to find the hidden platform nearly as well as the control animals, but show impaired performance after the position of the platform has been modified. As malperformance in the Morris water maze test has been associated with impaired long-term potentiation (LTP), electrophysiological studies were performed in hippocampal slices of Fmr1 knockout mice to check for the presence of LTP. Judged by field extracellular excitatory postsynaptic potential recordings in the CA1 hippocampal area, Fmr1 knockout mice express LTP to a similar extent as their wild type littermates during the first 1-2 hr after high frequency stimulation. Also, short-term potentiation (STP) was similar in both types of mice. To investigate whether Fmr1 is involved in the latter stages of LTP as an immediate early gene, we compared Fmr1 mRNA quantities on northern blots after chemical induction of seizures. A transient increase in the transcription of immediate early genes is thought to be essential for the maintenance of LTP. As no increase in Fmr1 mRNA could be detected, neither in cortex nor in total brain, during the first 2 1/2 hr after pentylenetetrazol-induced seizures, it is unlikely that Fmr1 is an immediate early gene in mice. In conclusion, we found no evidence for a function of FMR1 in STP or LTP.

The present study was aimed at examining the specificity of the action of heterotopic nociceptive conditioning stimulation (HNCS) by comparing its effects of those induced by a mental task (MT). Five test stimuli made from short CO2 laser pulses (duration: 40 msec; diameter: 10 mm; intensity: 0.25-0.8 Joules) were delivered every 30 to 45 sec at random to 4 different spots on the skin of the upper lip in 3 groups of 10 healthy subjects. The two most intense stimuli were perceived as painful, the two least intense stimuli as warm, and the intermediate stimulus as hot or near painful. Perception (VAS), reaction time (T) and cerebral evoked potentials (CEPs) were monitored before, during and after conditioning stimulation consisting either of HNCS (hand submerged in cold water) or of MT (arithmetic subtraction). Pain perception (first pain) threshold was increased in both conditioning stimulations; however, the stimulus-response curve and the neurophysiological correlates were differently affected. During HNCS, the stimulus-response curve was depressed and T was increased mainly for the intermediate stimulus, whilst CEP power density was reduced for all stimulus intensities; discrimination performance near pain threshold was dramatically depressed. During MT, the stimulus-response curve was shifted down toward higher stimulus intensities, T was equally increased for all stimulus intensities, whereas CEP power density was not changed; discrimination performance remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have used synthetic peptide analogs, corresponding to sequences within the pseudosubstrate domain ofprotein kinase C (PKC) or the autoregulatory domain of Caw+/calmoduln nt protein kinase II (CAMKI), in attempts to define the contribution of each of these protein kinass to induction of lo g-term potentiation (LTP). However, the specificity of these inhibitor peptides is not absolute. Using intraceliular delivery to rat CAl hippocampal neurons, we have determined the relative potency of two protein kin inhibitor peptides, and , as inhibitors of the induction of LTP. Both peptides blocked the induction of LTP; however, PKC-(19-36) was 30-fold more potent than Long-term potentiation (LTP) has been widely used as a cellular model of learning (1, 2). Among the biochemical mechanisms that contribute to this phenomenon, the activity of specific protein kinases appears to play a critical role (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Early studies demonstrated that extracellular application of nonselective protein kinase inhibitors, such as H-7, polymyxin B, or sphingosine, to hippocampal slice preparations could block induction of LTP (5-7). Subsequently, more compelling evidence that specific protein kinases were involved in the induction mechanism was provided by intracellular injection of various kinase inhibitors (8,9,11,14). Several of these studies examined the effect of synthetic peptide analogs modeled after autoinhibitory domains, which are present in second messenger-regulated protein kinases (20, 21). While these "pseudosubstrate" peptides were developed as selective protein kinase inhibitors (8, 9, 22-27), they have been found to exhibit multiple modes of action that vary with their exact structure. For example, peptides corresponding to different residues within the autoregulatory domain of Ca2+/calmodulin-dependent protein kinase II (CaMKII) can inhibit catalytic activity by one or more of the following mechanisms: (i) inhibiting the binding of ATP; (ii) inhibiting the binding of peptide or protein substrates; and (iii) directly binding to Ca2+/calmodulin, thereby functioning as a calmodulin antagonist (23)(24)(25)(26)(27).Based on the reported selectivity of several ofthese protein kinase inhibitor peptides, it has been suggested that protein kinase C (PKC) (14), CaMKII (8), or both (9) are required for induction of LTP. However, these studies did not examine in detail the relative potencies of the injected peptides for the blockade of LTP. Moreover, it is now apparent that the specificity of these pseudosubstrate inhibitor peptides is not absolute (19,22,28 Electrophysiology. Male Wistar rats (150-300 g) were anesthetized with diethyl ether and sacrificed by decapitation. The brain was removed and cooled in artificial cerebrospinal fluid (124 mM NaCl/2 mM KCI/1.25 mM KH2PO4/2 mM CaCl2/2 mM MgSO4/26 mM NaHCO3/10 mM glucose, pH 7.4, saturated with 95% 02/5% CO2 and kept at 2-40C) and the hippocampi were dissected out. Transverse slices (0.4 mm thick) were cu...