Neonatal Proinflammatory Stress Induces Accumulation of Corticosterone and Interleukin-6 in the Hippocampus of Juvenile Rats: Potential Mechanism of Synaptic Plasticity Impairments.

Infectious diseases in early postnatal ontogenesis can induce neuroinflammation, disrupt normal central nervous system development, and contribute to pathogenesis of cerebral pathologies in adults. To study long-term consequences of such early stress, we induced neonatal proinflammatory stress (NPS) by injecting bacterial lipopolysaccharide into rat pups on postnatal days 3 and 5 and then assessed the levels of corticosterone, proinflammatory cytokines and their mRNAs, and neurotrophins and their mRNAs in the hippocampus and neocortex of the one-month-old animals. Long-term potentiation (LTP) was studied in hippocampal slices as an index of synaptic plasticity. NPS-induced impairments of LTP were accompanied by the accumulation of corticosterone and IL-6 in the hippocampus. In the neocortex, a decrease in exon IV BDNF mRNA was detected. We suggest that excessive corticosterone delivery to hippocampal receptors and proinflammatory changes persisting during brain maturation are among the principal molecular mechanisms responsible for NPS-induced neuroplasticity impairments.

[Application of Paired-Pulse Stimulation for the Measurement of Inhibitory Transmission from the Hippocampal CA1 Field Potentials].

Short-term modifications of postsynaptic responses to paired-pulse stimulation depend not only on presynaptic facilitation. IPSP generated immediately after EPSP in response to the first stimulus is superimposed to synaptic facilitation, decreasing PPF at the shortest interpulse intervals or even producing paired-pulse depression. This effect can be used to measure the efficacy of inhibitory transmission specifically in the hippocampal CA1 area. Comparison of paired-pulse ratio was performed by different methods for the better extraction of GABA(A) IPSP involvement into CA1 field potentials of the rat hippocampal slices. Paired-pulse stimuli were delivered with the set of different stimulus intensity and two interpulse intervals--70 and 15 ms. PPF changes in accordance with exponential decrease of presynaptic residual calcium were observed mainly at low stimuli intensities, but more intensive activation suppressed PPF15 relative to PPF70, sometimes up to paired-pulse depression. The difference of the second in pair amplitudes (A2(15)-A2(70)) upon stimulus intensity corresponded to linear function with a negative slope. The negativity disappeared after bicuculline treatment, suggesting the involvement of GABA(A) inhibition. Therefore individual gradients of these functions can be considered as the coefficients of inhibition to measure its modifications in on-line regime.

Natural activation of caspase-3 is required for the development of operant behavior in postnatal ontogenesis.

We have previously demonstrated transient increases in caspase-3 activity in the hippocampus of rat pups from age 17 days. We report here our studies on the effects of inhibition of caspase-3 during this period on the acquisition of a two-way avoidance reaction. Rat pups received intracerebroventricular doses of the caspase-3 inhibitor Z-DEVD-FMK On postnatal day 18. Control animals of the same age received the inactive peptide Z-FA-FMK or isotonic saline solution. Inhibition of caspase-3 during the period of its natural activation in the hippocampus during early ontogenesis was found to impair the development of operant behavior in rats. This was apparent as a reduction in the efficiency of learning during acquisition of active avoidance reactions and decreases in the numbers of intersignal reactions. Administration of the inhibitor had no specific action on the types of conditioned reflex activity less associated with operant learning. Thus, there were no differences between the experimental and control groups in the numbers of emotional reactions to the conditioned stimulus. The number of orientational-investigative conditioned reactions also showed no change after administration of Z-DEVD-FMK. On the background of the reduction in the efficiency of the acquisition of the conditioned active avoidance reflex, the number of incomplete acts, in contrast to other types of conditioned reactions, increased significantly after administration of Z-DEVD-FMK, which is evidence for the persistence of the ability to form associative connections between activation of the conditioned signal and the need to move to the other sector. The difficulty in these animals arose at the decision-taking stage on choosing the appropriate form of behavior. Changes in orientational-investigative behavior were not associated with inhibition of caspase-3 during the critical period of development, as the effects of Z-DEVD-FMK and Z-FA-FMK were similar.

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.

Postnatal development of conditioned reflex behavior: comparison of the times of maturation of plastic processes in the rat hippocampus.

The formation of conditioned reflex fear, escape responses, and conditioned avoidance responses during acquisition of a conditioned two-way avoidance reflex was studied in rats of different ages. Rats aged 16-17 days acquired the conditioned reflex but not the escape reaction or the conditioned avoidance response; acquisition efficiency was higher than in adult rats. Escape responses appeared from postnatal day 18. The ability to acquire this type of learning was complete by age 3-4 weeks. Maturation of the mechanisms of the "classical" (the fear phase) and operant (transfer to another sector in response to the unconditioned stimulus) components did not facilitate acquisition of the conditioned two-way avoidance reflex until the middle of postnatal week 4. Learning efficiency in four-week-old rats was lower than in adults. It is suggested that the maturation of different types of memory may be associated with the periods at which plastic processes develop in the hippocampus.

Central administration of a caspase inhibitor impairs shuttle-box performance in rats.

Recent studies suggest that caspase-3-mediated mechanisms are essential for neuronal plasticity. N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val- Asp(OMe)-fluoromethyl ketone (z-DEVD-fmk), a caspase inhibitor with predominant specificity toward caspase-3, has been shown to block long-term potentiation in hippocampal slices. Intrahippocampal infusion of a caspase-3 inhibitor to rats has been shown to significantly impair spatial memory in the water maze. The present work was designed to study whether i.c.v. administration of a caspase-3 inhibitor z-DEVD-fmk impairs learning in other tasks related to specific forms of memory in rats. The rats received bilateral injections of z-DEVD-fmk or N-benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (z-FA-fmk) ("control" peptide) at a dose of 3 nmol. Administration of z-DEVD-fmk significantly decreased the number of avoidance reactions in some blocks of trials in the active avoidance (shuttle box) learning, while z-FA-fmk had no effect as compared with intact rats. However, only a slight effect of the caspase inhibitor across the session was found. z-DEVD-fmk impaired development of some essential components of the two-way active avoidance performance, such as escape reaction, conditioned fear reaction, and inter-trial crossings. Measurement of caspase-3 activity in rat brain regions involved in active avoidance learning revealed most expressed z-DEVD-fmk-related inhibition of the enzyme activity (about 30%) in the fronto-parietal cortex. A similar effect was close to significant in the hippocampus, but not in the other cerebral structures studied. In primary cultures of cerebellar neurons z-DEVD-fmk (2-50 microM) inhibited caspase-3 activity by 60-87%. We suggest that moderate inhibition of caspase-3 resulting from the central administration of z-DEVD-fmk to rats may impair active avoidance learning. Taking into account previous data on the involvement of neuronal caspase-3 in neuroplasticity phenomena we assume that the enzyme may be important for selected forms of learning.

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.

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.

Responses of hippocampal neurons at different stages of acquisition of conditioned reflex avoidance in rats.

The electrophysiological properties of hippocampal slices from trained and untrained rats presented with identical numbers of combinations during acquisition of a conditioned two-sided avoidance reaction were compared. Associative learning (presentation of combinations of conditioned and unconditioned stimuli) was accompanied by gradual increases in the amplitude of the population spike in hippocampal field CA1. However, achievement of the learning criterion was accompanied by a decrease in the amplitude of the population spike. The same type of difference between groups was seen in field CA1 using low-intensity stimulation of Schaffer collaterals, while there was no change in the amplitude at maximum-intensity stimulation. After the rats achieved the learning criterion, the level of facilitation in conditions of paired stimuli was significantly greater than in untrained animals and also as compared with the passive and active controls. These data provide evidence supporting the hypothesis that changes in the efficiency of synaptic transmission occur during the learning process. However, the direction of changes depends on the extent of training.

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.

Similarity of the effects of training and application of serotonin on the electrical activity of live hippocampal slices from rats.

A pronounced sensitization phase was observed on low-frequency stimulation of hippocampus slices from rats decapitated immediately after training to a conditioned bilateral escape reflex. The same effect was recorded in slices from control animals after brief application of serotonin. Serotonin had significantly smaller effect on the electrical activity of trained animals. Changes in the efficiency of the serotoninergic input to hippocampus neurons are suggested as one of the factors eliciting low-frequency facilitation in trained rats.

Frequency facilitation in the hippocampal slices of conditioned rats.

Electrophysiological parameters in the hippocampal slices of two experimental (prepared immediately (n = 27) and one week (n = 9) after conditioning) and two control (passive (n = 10) and active (n = 8)) groups of rats have been compared. The experimental rats were trained in a two-way avoidance chamber. The active control rats received the same number of conditioned and unconditioned stimuli without pairing. The threshold and amplitude of population spikes recorded from the CA1 pyramidal cell body layer to the stimulation of stratum radiatum and its modification in a train of 10 pulses (0.2 Hz) were measured. The mean values of the threshold intensity were not significantly different between any pair of these groups. The direction of the changes in the population spike amplitudes following pseudo-conditioning or conditioning was the same. However, the population spike amplitudes decreased more significantly in the slices from the conditioned rats. The increase in the frequency facilitation was specific for the slices of conditioned rats. The modifications in the mechanism of frequency facilitation in the reinforced pathways may represent an important mechanism for behavioural learning.