I. E. Kudryashov

Caspase activity is essential for long-term potentiation

Slices from rat hippocampus were incubated with the caspase‐3 inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethylketone (Z‐DEVD‐FMK) or with the inactive peptide N‐benzyloxycarbonyl‐Phe‐Ala fluoromethylketone (Z‐Phe‐Ala‐FMK) for 30 min. The peptides changed neither input–output curves nor paired‐pulse effects at 70‐msec interpulse intervals, nor amplitudes of pop spikes in the CA1 region 1.0–6.9 hr after the incubation. Slices taken 1.0–1.4 hr after Z‐DEVD‐FMK or inactive peptide treatment demonstrated similar long‐term potentiation (LTP) curves; however, LTP was suppressed significantly (P < 0.001) 1.5–3.4 hr after Z‐DEVD‐FMK treatment when compared to the corresponding inactive peptide group. LTP magnitude correlated with time after Z‐DEVD‐FMK (r = −0.74; P < 0.02) but did not depend on time after the inactive peptide treatment. After 3.5 hr, LTP was blocked completely. Z‐DEVD‐FMK did not have a significant effect on presynaptic function. The results are the first evidence that inhibition of caspase‐3 significantly decreases or fully blocks LTP in the CA1 region and suggest that caspase‐3 is essential for LTP. Candidate caspase‐3 substrates that may be cleaved for LTP induction and maintenance are discussed.

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.

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.

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.

Footshock stress alters early postnatal development of electrophysiological responses and caspase-3 activity in rat hippocampus

Postnatal changes in population spike (PS) amplitudes and caspase-3 activity were compared in the hippocampi of control rats and experimental animals subjected to a brief footshock on postnatal day (PD) 13. Footshock induced an increase in maximal PS amplitudes during the early period (from PD 14 to PD 16), however, the difference between stressed and control animals gradually decreased with age up to PD 21. No difference between hippocampal caspase-3 activity in control and footshock groups was revealed within the PD 14-17. However, caspase-3 activity in the latter group was significantly lower over the next period of postnatal development (PD 18-21). PS amplitudes in the slices of the footshock group significantly increased over PD 22-27. We suggest that footshock activates the development of hippocampal circuitry during early phases, this phenomenon mediating enhanced responsiveness as a result of an increased production of synaptic connections and related decrease in neuronal loss.

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.

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.

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.

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.

Regulation of cathepsin B and caspase-3 in long-term plasticity

It has been previously shown that high-frequency stimulation of Schaffer collaterals (HFS), which induces LTP in the CA1 area of rat hippocampus, under normal conditions results in the activation of caspase-3. In contrast to caspase-3, the activity of cathepsin B weakly decreased in hippocampal slices subjected to HFS (the significance level was p = 0.07). This effect was observed in all slices frozen for subsequent measurements of enzymatic activity in 15 minutes and 3 hours after LTP induction under normal conditions and in the presence of caspase-3 inhibitors. The reciprocal influence of HFS, which was observed in the control slices depending on their initial state (r = 0.59, p < 0.001 in control and r = 0.16, p = 0.46 in slices subjected to HFS), complicated simultaneous measurements of the activity of these enzymes. Though the effect of HFS on cathepsin B activity is not prominent, a significant difference in LTP magnitude in slices with low and high cathepsin B activity.