Effects of rat medial prefrontal cortex temporal inactivation on a delayed alternation task.

To determine the involvement of the medial prefrontal cortex (mPFC) in operant-type delayed alternation, microinjections of muscimol into the mPFC were used for temporal inactivation during behavioral tests in rats. The temporal mPFC inactivation showed effects related to both dorsal (decreased delay-dependent correct ratio, indicating working memory-related deficits) and ventral hippocampus inactivation (increased tendency to repeat errors) reported in our recent paper, without motor or sensory effects. These findings suggest that the mPFC integrates information from different hippocampal regions during a delayed alternation task.

Elevation of prefrontal acetylcholine is related to the extinction of learned behavior in rats.

To investigate whether changes occur in acetylcholine (ACh) levels in the rat medial prefrontal cortex (mPFC) during initial lever-press extinction training, in vivo microdialysis was used to measure mPFC ACh. Elevated ACh was found during this training period. Furthermore, this elevation significantly correlated with the number of responses found in the re-training session the next day, but not with that in the initial training. These results suggest that the mPFC ACh elevation during the initial training period enhances the progress of lever-press extinction across sessions.

Mouse hippocampo-prefrontal paired-pulse facilitation and long-term potentiation in vivo.

To confirm neural plasticity of the mouse hippocampo-prefrontal cortex (PFC) pathway, paired-pulse facilitation (PPF) and long-term potentiation (LTP) induction were determined in the pathway. In addition, we tested whether the plasticity differs in projections of the pathway from the dorsal (upper) and ventral (lower) parts of the temporal hippocampus. The results showed PPF and LTP of this pathway, and these differed between the projections. The projection from the upper part showed stronger PPF and weaker LTP compared with that from the lower part. These results suggest that the mouse hippocampo-PFC pathway is involved in learning and memory, and contains projections related to different functions.

Effect of long-term potentiation induction on gamma-band electroencephalograms in prefrontal cortex following stimulation of rat hippocampus in vivo.

We examined whether long-term potentiation (LTP) affects cortical gamma-band electroencephalograms (EEG) in the hippocampo-prefrontal cortex (PFC) pathway of anesthetized rats. The LTP induction increased the evoked PFC gamma-band EEG power (40-100 Hz) to 120-135% at 500-700 ms after test stimulation. A simple increment of stimulus intensity, instead of LTP induction, did not reveal this evoked increase. Neither LTP induction nor the intensity increment changed significantly the magnitude of an evoked decrease at around 100 ms or the spontaneous prestimulation gamma-band power. These results indicate that LTP in PFC specifically increases the evoked gamma-band EEG power, which may reflect a phasic mode of plastic neurotransmission through the hippocampo-PFC pathway in vivo.

Memory-related acetylcholine efflux from rat prefrontal cortex and hippocampus: a microdialysis study.

To investigate the relationship between the prefrontal and hippocampal acetylcholine (ACh) systems and working memory, an in vivo microdialysis study was conducted. A group of rats was trained to perform a working memory task, delayed alternation, in an operant chamber for food reinforcement. The rats had to choose one of two response levers in an alternative manner in each trial, with a certain interval between trials. They had to remember which lever they chose in the previous trial without the assistance of external cues. Another group was trained to perform a reference memory task, cued alternation, in which the behavioral sequence was identical, but an external cue was provided. After stable behavior was established, a dialysis probe was implanted into the prefrontal cortex or the hippocampus of each rat. The extracellular concentration of ACh in the dialysates from the prefrontal cortex increased during performance of the delayed alternation task, while the hippocampal ACh showed a more distinct increase during performance of the cued alternation task. These results suggest that the prefrontal ACh is mainly related to working memory, whereas the hippocampal ACh is mainly related to reference memory.

Effects of rat ventral and dorsal hippocampus temporal inactivation on delayed alternation task.

To determine the involvement of the hippocampal regions in a operant-type delayed alternation task of short delay or long delay, microinjections of muscimol into the hippocampus were used for temporal inactivation during the behavioral test in each rat. Dorsal hippocampal inactivation impaired the correct ratio of long delay. Ventral hippocampal inactivation showed no changes in the correct ratio, however, it increased the tendency of perseveration in long delay. These findings suggest hippocampus has regional differentiation in delayed alternation task.

Differences in paired-pulse facilitation and long-term potentiation between dorsal and ventral CA1 regions in anesthetized rats.

To clarify hippocampal regional differences in synaptic plasticity, paired-pulse facilitation (PPF, a form of short-term plasticity), long-term potentiation (LTP, a form of long-term plasticity), and their interactions were studied in the dorsal and ventral hippocampal CA1 regions of anesthetized rats. Baseline PPF and post-LTP PPF experiments were conducted at interstimulus intervals (ISIs) of 20-320 ms. A general protocol (100 Hz, 1 s) and a stronger protocol (250-Hz pulse series) were applied for LTP induction. PPF were observed in both regions; however, the degree was lower and the range of ISIs was narrower in the ventral region compared with the dorsal region. The degree of ventral LTP was lower than that of the dorsal LTP. The interaction between PPF and LTP was observed in both regions (PPF change correlated inversely with degree of baseline PPF). However, this was also different in each region. Dorsal PPF increased or decreased; in contrast, ventral PPF of short ISIs after LTP only decreased. These regional differences in short-term and long-term synaptic plasticity may explain a consequence of different afferent inputs and information processing.

Comparative induction of long-term depression between dorsal and ventral hippocampal CA1 in the anesthetized rat.

We studied whether a protocol reported as in vivo prefrontal long-term depression (LTD) induction protocol, also induced LTD in the anesthetized rat hippocampal CA1, and whether differences in LTD induction existed between dorsal and ventral CA1, by low-frequency stimulation (LFS) (1 Hz, 900) or low-frequency burst stimulation (LFBS) (5-pulse burst at 4 ms interpulse intervals at 1 Hz, 900), hippocampo-prefrontal LTD induction protocol. Though LFS failed to induce stable LTD in dorsal or ventral CA1, LFBS reliably induced LTD in the ventral not dorsal CA1. This similarity between ventral hippocampal and hippocampo-prefrontal LTD induction thus implies their serial integration process, ventral CA3-CA1-prefrontal cortex pathway and observed dorsal and ventral differences involved in behavioral functions such as learning.

Disturbance of rat lever-press learning by hippocampo-prefrontal disconnection.

To determine whether the medial prefrontal cortex (PFC), ventral hippocampus and hippocampo-PFC pathway are involved in operant lever-press learning, we conducted lidocaine injections to these brain sites. Rats were injected immediately after lever-press acquisition in the first training, and the second 5-min test the next day. Results showed the response rate of either PFC- or ventral hippocampus-inactivated rats to be lower than that of control rats in the test the next day. Rats having lidocaine injected into the unilateral ventral hippocampus combined with contralateral medial PFC also showed lower response rate in their tests. These results suggest that hippocampo-PFC disconnection disturbs operant learning.

Induction of stable long-term depression in vivo in the hippocampal-prefrontal cortex pathway.

We studied excitatory field potentials in the medial prefrontal cortex (mPFC, prelimbic area) to electrostimulation of the ventral hippocampus (CA1/subicular region) in the anaesthetized rat. Nine hundred stimulus trains (5 pulses at 250 Hz) applied at 1 Hz to the ventral hippocampus significantly and persistently depressed the amplitude and maximal slope ( approximately 55% for each index) of the prelimbic field potentials, but did not change the latency of the maximal slope or peak negativity. Twelve stimulus trains (50 pulses at 250 Hz) applied subsequently at 0.1 Hz restored the depression back to control level, and this reversible depression was maintained for at least 13 h. Cumulative depressive effects on the prelimbic field potential amplitude and maximal slope were observed upon addition of stimulus trains in the hippocampus. An important implication of the results is that the direct pathway from the hippocampus to the mPFC in the rat retains long-term depression (LTD) as a neuroplastic form in vivo. This form could cooperate with long-term potentiation (LTP) and such a bi-directional synaptic plasticity in the prefrontal cortex contributes to how cortical neural networks store information.

Enhancement by 1-oleoyl-2-docosahexaenoyl phosphatidylcholine of long-term potentiation in the rat hippocampal CA1 region.

We reported previously that administration of 1-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylcholine (ODHPC), a kind of phosphatidylcholine, enhanced discriminatory shock avoidance learning in rats. Since long-term potentiation (LTP) of the hippocampus has been suggested to be a physiological substrate of some forms of memory, we investigated the effects of ODHPC on LTP in the rat hippocampal CA1 region. LTP in the amplitude of population spikes in the CA1 region was induced by tetanic stimulation in anesthetized rats. ODHPC significantly increased magnitudes of LTP in a dose-dependent manner when injected intraperitoneally 20 min before inducing LTP. However, administration of 1,2-dioleoyl-sn-glycero-3-phosphorylcholine, in which only docosahexaenoyl residue of ODHPC was replaced with oleoyl residue, did not affect LTP. These results suggest that ODHPC enhances hippocampal LTP by its specific conformation.

Dopamine and acetylcholine elevation on lever-press acquisition in rat prefrontal cortex.

To determine whether the rat medial prefrontal cortex (PFC) is involved in acquiring operant learning, we observed changes in extracellular concentration of dopamine (DA) and acetylcholine (ACh) in the rat medial PFC during lever-press acquisition (acquisition group) or retrieval (retention group) using in vivo microdialysis. We found that DA or ACh elevation related to acquisition occurred. DA elevation was observed in the acquisition group only. These results indicate that the medical PFC is related to acquisition, and suggest that interaction between DA and ACh may be involved in learning acquisition.

Effect of active oxygen radicals on protein and carbohydrate moieties of recombinant human erythropoietin.

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radicals generated from a Fenton system in the presence of 0.016 mM H2O2, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H2O2). The tryptophan destruction was detected at 0.016 mM H2O2 and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increased of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.

Long-term potentiation in the rat hippocampal CA1 region is inhibited selectively at the acquisition stage of discriminatory avoidance learning.

Long-term potentiation (LTP) of hippocampal synaptic efficacy has been regarded as a synaptic model of learning and memory. We examined whether the induction of LTP in the hippocampal regions was altered with the advance of discriminatory avoidance learning. Evoked potentials in the CA1 region or dentate gyrus were recorded before and after tetanic stimulation in anesthetized rats which had been given training sessions 24 h before. LTP of the amplitude of population spikes and the slope of excitatory postsynaptic potentials recorded in the CA1 region was smaller in rats at a learning stage 24 h after the avoidance rate had first been significantly increased than in naive rats. The magnitude of LTP was not altered in rats which had been exposed to the first training session or in those which had received overtraining. The inhibition of LTP in the CA1 was neither due to stress accompanied with training nor liberation from the stress by learning avoidance response. In contrast, LTP induced in the dentate gyrus was rather enhanced at the learning stage when LTP in the CA1 was inhibited. The results suggest that the acquisition of discriminatory avoidance learning selectively inhibits LTP in the hippocampal CA1 region.

The loss of in vivo activity of recombinant human erythropoietin by active oxygen species.

The effects of active oxygen species on the in vivo activity of recombinant human erythropoietin (EPO) treated by Fenton system, xanthine (X) plus xanthine oxidase (XO) system and hydrogen peroxide (H2O2) has been studied by means of counting the increase in number of hemolyser-resistant cells (HRCs) in EPO-injected mice. The results showed that both Fenton and X plus XO systems caused a significant reduction of the activity in proportion to the concentration of generated active oxygen species. Meanwhile, the treatment of EPO with H2O2 alone resulted in a relatively slight reduction of the activity. Electrophoretic studies on the structure of EPO revealed that its main protein band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) disappeared in proportion with the extent of exposure to active oxygen generating systems. Both Fenton and X plus XO systems caused a significant loss of fluorescence in the pyridylamino (PA-) sugar chain in proportion to the concentration of generated active oxygen species, and no degradation products in the sugar chain part of the PA-sugar chain were detected. This showed that aromatic groups in EPO were sensitive to attack by active oxygen species. These results provide evidence that hydroxyl radical and other active oxygen species have a potential to react with EPO, leading to a reduction of its in vivo activity.

Intraperitoneal injection of 1-oleoyl-2-docosahexaenoyl phosphatidylcholine enhances discriminatory shock avoidance learning in rats.

Effects of injection of 1-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylcholine (ODHPC) on learning ability were investigated in rats using discriminatory shock avoidance learning task. When ODHPC (2 mumol) was intraperitonealy administered 5 min before the beginning of the first trial of learning task from the second to fifth sessions, avoiding rates of the ODHPC-injected group were significantly higher than those of the control group. However, any injection of ODHPC derivatives, such as 1-oleoyl-2-docosahexaenoyl-diacylglycerol, 1,2-dioleoyl-sn-glycero-3-phosphorylcholine, glycerophosphorylcholine, docosahexaenoate, oleate and choline chloride, did not affect learning. These results suggest that intraperitoneal ODHPC injection enhances learning ability by its specific conformation.

1-Oleoyl-2-docosahexaenoyl phosphatidylcholine increased paradoxical sleep in F344 rats.

The effects of administration of 1-oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylcholine (O-DH-PC), a kind of lecithin, and of glycerophosphorylcholine (GPC) on sleep were investigated in male F344 rats. Intracerebroventricular administration of O-DH-PC at a dose of 10 micrograms/rat induced significant increase in paradoxical sleep time and total sleep time in the following 24 h, while administration of GPC did not. Results suggest that O-DH-PC affects on neuronal mechanism relating to paradoxical sleep, and that the effect of O-DH-PC might be caused by fatty acid residues, rather than choline residue.

Effect of ingestion of thermally oxidized frying oil on peroxidative criteria in rats.

Thermally oxidized rapeseed oils (4 levels of deterioration; used by a manufacturer of fried fish paste in a conventional manner) were fed to rats at a practical level of concentration. Rats were fed a diet ad libitum for 13 weeks that contained 15% of a test oil. The effects of the diet on several biochemical criteria related to peroxidative alterations were investigated. In groups given thermally oxidized oils relative liver weight, relative kidney weight, thiobarbituric acid-reactive substances (TBA-RS) in the liver and reduced glutathione content were increased significantly in proportion to the degree of deterioration of the oil, compared with the group given fresh oil. Tocopherol contents in both serum and liver were decreased considerably in proportion to the deterioration level of the supplied oils. The above criteria correlated well with various deterioration indices of the oil. For instance, TBA-RS was well correlated (p less than 0.001) with petroleum ether-insoluble oxidized fatty acid (r = 0.9191), column chromatographically separated polar fraction (r = 0.9056), glyceride dimer fraction (r = 0.9023) and carbonyl value (r = 0.8647).