Caspase-3 activity in hippocampal slices reflects changes in synaptic plasticity.

Electrophysiological measures of the functional activity of neurons in field CA1 in conditions of paired-pulse stimulation of Schäffer collaterals were performed in relation to the involvement of caspase-3 in mediating neuroplasticity; the relationship between functional activity and caspase-3 activity in hippocampal slices from Wistar rats was addressed. Enzyme activity was assessed in each individual slice at the end of the electrophysiological experiment. The results obtained here showed that the highest level of enzyme activity was seen when the efficiency of interneuronal interactions decreased. Nerve cell excitability showed no changes; interactions increasing synaptic efficiency, particularly in paired-pulse stimulation, produced normal response amplitudes. Further deterioration of the functional state of slices and impairments in spike generation were accompanied by increases in caspase-3 activity to the normal level. Increases in the activity of another proteinase, cathepsin B, were generally seen in any deviation from normal functioning, though there was no correlation with any of the electrophysiological parameters. It is suggested that high caspase-3 activity in slices is linked with neuroplastic processes in synapses and has no direct relationship to nerve cell apoptosis.

Kindling in the early postnatal period: Effects on the dynamics of age-related changes in electrophysiological characteristics of hippocampal neurons.

The effects of chronic administration of pentylenetetrazole (PTZ) during early ontogenesis (from postnatal day 14) on the dynamics of age-related changes in electrophysiological characteristics of rat hippocampal slices were studied. Unlike the situation in adult animals, convulsive activity did not develop in rat pups in response to repeated injections. Comparison of the amplitude characteristics of total monosynaptic responses of neurons in hippocampal field CA1 to application of single and paired (separated by 70 msec) stimulation of Schäffer collaterals during the period of maximally intense hippocampal synaptogenesis (at weeks 2-3 of postnatal development) revealed no significant differences between the control group (administration of isotonic saline) and the group given PTZ. The level of suppression of facilitation in paired-pulse stimulation with a short interstimulus interval (15 msec) was significantly less in hippocampal slices from rat pups from the PTZ group. However, as compared with the passive control, the direction of rearrangements in the two experimental group was essentially the same. Nonetheless, regular administration of PTZ during the period of maximally intense hippocampal maturation affected the development of its characteristics. This was not only apparent as a deficiency of inhibitory processes. Increases in the intensity of test stimuli applied to hippocampal slices from PTZ-treated rat pups at 27-48 days of age led to relatively lower response amplitudes as compared with those seen in hippocampal slices from control (given isotonic saline) rats of the same age. The level of facilitation in paired-pulse stimulation with an interstimulus interval of 70 msec showed no difference, decreasing to similar extents in both groups as compared with the passive control group. In addition, hippocampal slices from the PTZ group showed significant decreases in the magnitude of long-term potentiation. Changes occurring in the hippocampus after regular administration of PTZ did not correlate with the development of convulsive activity. The only significant relationship involving the intensity of convulsions was with the increase (compared with the normal for age) in the amplitudes of responses to minimal-intensity test stimuli.

Long-term potentiation in the hippocampus in conditions of inhibition of caspase-3: analysis of facilitation in paired-pulse stimulation.

Treatment of hippocampal slices with the caspase-3 inhibitor Z-DEVD-FMK led to a decrease in the magnitude of long-term potentiation (LTP), which developed over time. Testing with paired stimuli separated by an interval of 70 msec showed that after caspase-3 inhibition, as compared with control slices, the second response in the pair showed no increase in amplitude in conditions of LTP. In these conditions, the magnitude of LTP depended on differences in the amplitudes of the first and second responses before induction of LTP. LTP was absent in slices with initially highly efficient afferent stimulation and correspondingly low levels of facilitation in paired-pulse stimulation. It is suggested that inhibition of caspase-3 prevents the structural rearrangements in LTP associated with the involvement of new synapses and neurons in the response.

Identification of the diamond-like B-C phase by confocal Raman spectroscopy.

The new diamond-like B-C phase was obtained from the graphite-like BC phase in a laser-heated diamond anvil cell at high temperature 2230+/-140 K and high pressure 45 GPa. Raman spectra of the new phase measured at ambient conditions revealed a peak at 1315 cm(-1), which was attributed to longitudinal-optical (LO) mode. The X-Y Raman mapping was used to investigate spatial distribution of the diamond-like phases and was shown to be a powerful tool in studying the sp(2)-to-sp(3) phase transformations occurring in the diamond cell under high temperature and high pressure.

Age-related changes in the characteristics of evoked responses in field CA1 in rat hippocampus slices after deafferentation of the forelimb.

The effects of in situ transection of the median nerve in baby rats on the formation of the properties of synaptic transmission in the hippocampus were studied. Paired-pulse stimulation was used to identify the characteristics of short-term facilitation in living slices. Significant deviations in the time dynamics of facilitation from control values were seen in operated animals. Analysis showed that the decrease in the population spike amplitude, which we have described previously, may be due not only to functional causes, but also to morphological causes--elimination of neurons and their processes. It is suggested that a period of destructive changes is followed by the onset of a phase of intense formation of new synaptic contacts. The nature of the heterochrony of electrophysiological measures does not lead to any conclusion as to whether the maturation of intrahippocampal systems leads or lags in the experimental baby rats. It is suggested that hippocampal development along a new, abnormal pathway is induced.

Raman microspectroscopic study on polymerization and degradation processes of a diacetylene derivative at surface enhanced Raman scattering active substrates. 1. Reaction kinetics.

Raman microspectroscopy was applied to study the polymerization kinetics of the Langmuir-Blodgett (LB) films of 10,12-pentacosadiynoic acid (DA) adsorbed on surface enhanced Raman scattering (SERS) active Ag island films. A two-dimensional (2D) Raman microscopic image measured at 1520 cm(-1) exhibits bright and dim spots with the diameter of several hundred nanometers. Raman microscopic spectra, measured by defocusing the excitation laser light (532 nm, diameter of ca. 10 mum) on the samples at room temperature, proved the occurrence of the surface processes consisting of the formation of polydiacetylene (PDA) in the blue phase, its conversion to the red phase, and subsequent bleaching. These reactions were negligible under the same condition for the DA-LB films prepared on a smooth (i.e., SERS-inactive) Ag film, indicating that the 532-nm-induced polymerization and the bleaching process are enhanced by the SERS-active substrates. At low temperatures below -50 degrees C, the Raman microscopic measurements proved the formation of the blue phase and its conversion to the red phase with much lower reaction rates compared to the corresponding rates at room temperature. The bleaching, however, was much suppressed at the low temperatures. The kinetic analyses of the formation of the blue phase and its conversion to the red phase were performed by using the intensity changes of the Raman bands due to the blue (1477 cm(-1)) and red (1517 cm(-1)) phases as a function of the irradiation time. The results strongly suggested the existence of at least two processes taking place simultaneously on the SERS-active substrates; that is, one of the processes is a sequential reaction, DA-monomers --> PDA in the blue phase --> PDA in the red phase, and the other is another sequential reaction, DA-monomers --> PDA in the red phase --> degradation species (probably amorphous carbon). Thus, even at the low temperatures, there occurs the surface reaction consisting of the formation of PDA and its degradation. The reaction can be ascribed to a process taking place at the highly SERS-active site, which gives the bright spot (so-called "hot spot") on the 2D Raman image, as proved by the confocal Raman microscopic measurement in the following paper.

Raman microspectroscopic study on polymerization and degradation processes of a diacetylene derivative at surface enhanced Raman scattering active substrates. 2. Confocal Raman microscopic observation of polydiacetylene adsorbed on active sites.

Confocal Raman microscopic measurements were performed at room temperature on the Langmuir-Blodgett (LB) monolayer of 10,12-pentacosadiynoic acid (DA) prepared on surface enhanced Raman scattering (SERS) active Ag island films, two-dimensional (2D) Raman images of which exhibit bright and dim spots on a dark background. The measurements performed by focusing the excitation laser light (488 nm) on the dark background indicate the prompt appearance of the Raman bands (1515 and 2115 cm(-1)) due to polydiacetylene (PDA) in the red phase and subsequent diminution of the Raman bands. On the other hand, the spectra observed by focusing the excitation laser spot on the dim and bright spots exhibit almost random fluctuations, giving rather narrow Raman bands in the 1620-1000 cm(-1) region, which appear and disappear temporarily with varying intensities under the continuous irradiation at 488 nm. Broad Raman bands appear around 1580 and 1360 cm(-1), which are ascribable to amorphous carbon, at a later stage of the observation, the intensities from the bright spot being more than 100 times stronger than those from the dim spot. The narrow bands are ascribed to a series of carbonaceous intermediates such as polyenes, graphite sheets with various sizes, and folded or reorganized forms of the sheets including carbon nanotubes and fullerenes, which are formed during the conversion of PDA to amorphous carbon. The random spectral fluctuation was interpreted by considering that the intermediates undergo thermally activated diffusion and get temporarily in contact with the SERS-active site, resulting in the enhancement of their Raman bands and the fluctuation.

Inhibition of caspase-3 blocks long-term potentiation in hippocampal slices.

Incubation of rat hippocampal slices with the specific caspase-3 inhibitor Z-DEVD-FMK led to suppression of long-term potentiation (LTP), which developed over a period of time. Incubation of Z-DEVD-FMK for 4 h and more prevented tetanization from evoking LTP; Z-DEVD-FMK had no effect on baseline measures of synaptic plasticity and short-term plasticity (population spike amplitude, level of facilitation in conditions of paired stimuli). These data provide the first evidence for the involvement of a mechanism mediated by caspase-3 in the phenomenon of LTP.

Periods of postnatal maturation of hippocampus: synaptic modifications and neuronal disconnection.

The paired-pulse paradigm was used to study the maturation of CA1 population spikes (PS) in the hippocampal slices of Wistar rats. Measurements were taken daily, from postnatal day (PN) 14 to PN27. In the slices from younger animals, inputs exhibit strong paired-pulse profile, which may be associated with low synaptic efficacy. Both responses increased during the third week of life, however, PS1 increased faster so that the PS1/PS2 ratio increased during the early period and remained increased thereafter. This may reflect postnatal modifications of synaptic transmission mediating the increase in hippocampal responses. Modifications of synaptic efficacy are prevailing during early phases while other mechanisms take over at later stages. Partial correlation analysis suggests that the decline of PS amplitude after PN19 may be due to the decrease in the number of connected neurons rather than to modifications of the synaptic efficacy. Thus, the actual direction and magnitude of postnatal PS maturation is suggested to depend on the balance of these two factors. The transient decline of PS amplitude coincided with a period of caspase-3 activation. There was a clear general trend for caspase-3 activity to decrease before PN17, while the inverse trend was observed during next period up to PN21.

The effects of forelimb deafferentation on the post-natal development of synaptic plasticity in the hippocampus.

The effects of partial deafferentation of the forelimb on the development of long-term potentiation in the hippocampus of rats aged 13-18 days were studied. Long-term potentiation in hippocampus field CA1 was of greater amplitude and duration in control rats at 16-18 days of post-natal ontogenesis than in adult animals. Partial deafferentation by section of the median nerve in the forelimb on the 13th day of life led to the disappearance of this excess at 16-18 days. The peak in synaptic plasticity occurred later in operated animals--on day 17--and was much less marked than in controls. The decreases in the amplitude and duration of long-term potentiation in hippocampal field CA1 in operated animals provides evidence for a decrease in the sensitivity and/or number of NMDA receptors. This suggests that partial deafferentation of one limb may lead not to a decrease but to an increase in spike and synaptic activity in the hippocampus, which in normal conditions may affect the maturation of the plastic properties of synaptic transmission associated with the expression and positions of NMDA receptors. The level of long-term potentiation in sham-operated rats was significantly greater than in controls of the same age. This significant increase in NMDA-dependent long-term potentiation may be explained by a decrease in the level of activation due to anesthesia. It is suggested that the decrease in the spike activity of cells receiving signals from the median nerve may be compensated for by activation of other specific and non-specific inputs.

Ontogeny of synaptic transmission in the rat hippocampus.

Electrophysiological characteristics of the hippocampal slices of juvenile (14-27 days) or young (36-40 days) Wistar rats have been compared. In the juvenile rats measurements were taken daily, from postnatal day (PN) 14 to PN27. Input-output curves were used to quantify the ontogeny of excitatory processes. The dynamic of population spike (PS) maturation was not even during the investigated postnatal period. After day 19 transient decrease of PS amplitude was observed until day 22. There were also some differences between the shape of input-output curves from the slices of rats of different ages. In general, PS was saturated at lower intensities in younger animals. The slices from 19-day-old rats did not display saturated input-output curve with 2-20 V stimuli intensities. But input-output curves on PN20-22 were rather similar to that obtained before PN19. The periods of gradual increase and subsequent decrease of PS amplitudes during early ontogeny correlate with the appearance of certain forms of behaviour. This fact suggests that hippocampal PS amplitude depression may be relevant functionally.

The effects of vasopressin on hippocampal slices from trained rats.

The effects of vasopressin on hippocampal slices from control and pretrained rats were compared. Prolonged presentation of both combined and noncombined conditioned stimuli (CS) and unconditioned stimuli (US) increases the sensitivity of hippocampal neurons to vasopressin. The effect of this was that responses to application of DG-AVP after training and active control treatment was biphasic, consisting of initial excitation and subsequent inhibition; similar effects were seen in control slices when the peptide concentration was increased. Increases in the excitatory phase occurred with presentation of the CS and US and were independent of the training factor. The inhibitory phase increased to a greater extent on presentation of combinations of the CS and US. It is suggested that increases in depression in response to application of vasopressin in trained animals, as compared with active controls, are due to a higher level of free intracellular calcium occurring as a result of the training procedure.

Ribonuclease improves the state of hippocampal sections in the post-ischemic period.

Living hippocampal slices from Wistar rats were used to study the dynamics of changes in population electrical responses in field CA1 to electrical stimulation of Shaffer collaterals during the development of ischemia (imposed by exclusion of oxygen and glucose from the perfusion solution). These studies showed that during ischemia, addition of ribonuclease (a blocker of protein synthesis) to the perfusion solution resulted in a significantly smaller increase in the latent period of the response and slowed the onset of the reduction in the amplitude of the evoked potential, and promoted faster recovery of the response after the ischemia session ended. It is suggested that the reduction in protein synthesis due to ribonuclease preserved energy reserves in the nerve tissue, which in turn promoted more complete recovery of neuron function in the post-ischemic period.

Elaboration of a conditioned reflex in a single experiment with simultaneous recording of neural activity.

In 18 of 20 experiments with click CS, and in 5 of 10 experiments with flash CS, we have elaborated a conditioned EMG response in a single session (less than 60 pairings). The US was direct electrical stimulation of the rabbit's motor cortex that evoked a movement of the forelimb. Electrical stimulation of the lateral hypothalamus was used as reinforcement. Parameters for this reinforcement were chosen to evoke a feeding reaction or self-stimulation in the freely behaving animal. The elaborated EMG response satisfied most of the required characteristics of a conditioned reflex. These included spontaneous recovery after extinction, savings of long duration, specificity to stimulus pairing, and weak efferent and afferent generalization. In most experiments with click CS, the elaborated phasic response had an amplitude of 0.1-6 mV with a latency of 12-16 msec. In form and latency the conditioned response was similar to the unconditioned startle reaction of the same animal under chloralose anesthesia, or to its unanesthetized response to a loud sound. However, the conditioned response differed from the startle reaction in that it was localized. Extracellular recordings of 2-5 neurons were simultaneously made from sensory motor cortex near the point at which the US was applied. In 7 experiments 17 neurons were followed through the entire cycle of elaboration and extinction of the conditioned response. Seven neurons showed a statistically significant (P less than 0.05) increase of the response to CS during conditioning. Latencies were 20-140 msec. Interactions between neurons were studied by computing cross-correlograms and joint PST scatter diagrams. These measures were less informative than we had hoped because of the low level of spontaneous and evoked activity, and because of the small numbers of stimulus presentations that were needed for elaboration and extinction of the conditioned reflex. In isolated presentations of the CS after elaboration of the CR, we sometimes observed neural responses to click with a latency less than 6 msec. We propose that at least one of the pathways involved in the localized conditioned startle reflex reported here goes through the sensory motor cortex.