Early effects on restoration of evoked field potentials in the hippocampal CA(1) region after reversible hypoxia/hypoglycemia by the radical scavenger N-tert.-butyl-alpha-phenylnitron.

In transverse hippocampus slices a short period of hypoxia/hypoglycemia induced by perfusion with an O(2)/glucose-free medium caused early loss and incomplete restoration of evoked field potentials in the CA(1) region. In the present study a search was made for whether the formation of free oxoradicals immediately after starting the hypoxic phase could be part of the breakdown and incomplete restoration of the excitatory potentials (EPs). It was shown that preincubation and postischemic incubation with the radical scavenger PBN did not prevent the potential breakdown but significantly enhances potential restoration, even when PBN was added to the perfusion medium 40 min after hypoxia. Thus, free oxoradicals may damage membrane constituents such as receptors or channel proteins at a very early phase, before neuronal death is pronounced. The results also show that treatment with radical scavengers has a beneficial effect on early hypoxic damage.

Conditioning hypoxia in vivo alters neuronal excitability of hippocampal neurones of rats in vitro and in vivo.

PURPOSE: Moderate normobaric hypoxia causes long-lasting protection against damage induced by a second ischemic or traumatic insult. The changes induced by such conditioning hypoxia are not yet fully understood. The protective effect has been described in various publications but other effects seem possible. METHODS: In order to search for effects of conditioning normobaric hypoxia yet unknown, we measured O-Mg++ potentiation and classical long-term potentiation (LTP) in the CA, region of hippocampal slices seven days after the conditioning procedure. Furthermore, tests were also carried out in chronically implanted rats as to whether LTP was changed in the dentate gyrus after conditioning hypoxia. RESULTS: In slices taken from animals which received conditioning hypoxia, O-Mg++ potentiation and classical LTP of the population spike were significantly enhanced, when compared to population in two control groups. Anoxic LTP, i.e. increase in the population spike of the field potential in the dentate gyrus immediately after finishing the procedure, and a tendency to elevation of LTP seven days after conditioning were also found in chronically implanted animals which were subjected to conditioning hypoxia. CONCLUSIONS: lt. may be hypothesized that elevated Ca++ transport in the neurons during conditioning hypoxia is responsible for both long- lasting increases in plastic reactions and the protective effect.

Moderate hypoxia reduces pentylenetetrazol-induced seizures.

Mice were exposed to an atmosphere consisting of 7% O2 and 93% N2 or 5.5% O2 and 94.5% N2 for 60 min. The susceptibility of the mice to the convulsive effect of pentylenetetrazol (PTZ) was decreased, in comparison to that of naive or sham-exposed controls, 1 and 7 days after exposure to 7% O2. A significant protective effect against PTZ-induced seizures was not observed in mice exposed to 5.5% O2. N-methyl-D-aspartate (NMDA) administrated immediately after exposure to the hypoxic atmosphere, had no significant influence on the protective effect of hypoxia. Treatment of naive or sham-exposed mice with NMDA resulted in protection against PTZ-induced seizures when they were tested 7 days later. Dizolcipine (MK 801), at a dose of 0.01 mg/kg injected i.p. 10 min before hypoxia, abolished the protective effect of hypoxia; higher doses (0.1 or 0.3 mg/kg) of MK 801 were not effective. The adenosine A1 receptor antagonist 1,3-diethyl-8-phenylxanthine (DPX), administered at a dose of 0.1 mg/kg s.c. before hypoxia, blocked the decrease in the susceptibility to the convulsive effect of PTZ. DPX also blocked the protective effect, seen after 7 days, of NMDA given to control mice. These results suggest that both NMDA and adenosine A1 receptor-mediated processes were involved in the protective effect of moderate hypoxia against PTZ-evoked seizure.

Phe1-substituted beta-casomorphin-5 analogues with analgesic activity.

The antinociceptive potency of linear and cyclic beta-casomorphin-5 (CM-5) analogues, modified in position 1 by substitution of the tyrosine (Tyr) by the phenylalanine (Phe) residue, was studied using the vocalization test. With the exception of the linear [Phe1,D-Orn2]CM-5, the Phe1-substituted linear and cyclic casomorphin analogues exhibit remarkable analgesic potency compared to morphine, although the opioid receptor affinity and the opioid activity in vitro is diminished compared to the corresponding Tyr-containing analogues. The analgesic effect of the compounds is mediated by activation of opioid receptors, because it can be antagonized with naloxone. Furthermore, it was demonstrated that the [Phe1,D-Orn2,D-Pro4]CM-5, which was about sixfold more potent than morphine, developed cross-tolerance to morphine.

Influence of beta-casomorphins on apomorphine-induced hyperlocomotion.

Derivatives of beta-casomorphin Tyr-Pro-Phe-Pro-Gly and their des-Tyr1-derivatives were investigated on the model of apomorphine-induced hyperlocomotion (1 mg/kg = 3 microM/kg, IP). D-Pip4 CM 5 (5 nM) inhibited the apomorphine hypermotility completely, while D-Phe3 CM 5 (5 nM) and D-Pro4 CM 5 (5 nM) decreased it only to about 50%. The normal exploration was nearly completely inhibited by D-Pro4 CM 5 (40 nM), by D-Pip4 CM 5 (5 nM) depressed to 20%, and by D-Phe3 CM 5 (10 nM) to 35%. The maximum inhibition of apomorphine-induced hyperlocomotion by the des-Tyr-casomorphin derivatives was about 50%. The dose-response curves were U-shaped. The exploratory activity was not significantly influenced. The mode of action and the involvement of different neurotransmitter systems in the inhibitory effect of beta-casomorphin derivatives on apomorphine hyperlocomotion are discussed.

Adrenalectomy attenuates the improvement of memory in rats by peripheral application of Des-Tyr-D-Pro4-casomorphin.

beta-Casomorphin derivatives without the N-terminal amino acid tyrosine possess memory-improving effects after central and peripheral application. We investigated the significance of adrenal glands for the memory improving effect of the systemically applied beta-casomorphin derivative des-Tyr-D-Pro4CM (Pro-Phe-D-Pro-Gly) in a learning experiment. Seven-week-old rats were adrenalectomized or sham operated. One week after surgery the rats were trained in an active avoidance task in a shuttle box. Five avoidance reactions were taken as learning criterion. After training 10 nmol/kg des-Tyr-D-Pro4CM or saline (10 ml/kg) was subcutaneously applied. There were no differences in acquisition between adrenalectomized and sham-operated rats. The memory retention of sham-operated animals was improved by des-Tyr-D-Pro4CM. In adrenalectomized rats this positive effect could not be observed. The involvement of adrenal glands in the peptide effect during learning and retention is discussed.

Linear and cyclic beta-casomorphin analogues with high analgesic activity.

We investigated the antinociceptive efficacy of casomorphin (CM) derivatives using the vocalization test. Male Wistar rats received chronic microcannulae into the right lateral ventricle. One week later we examined the analgesic effect of CM derivatives 10, 30, 60, and 90 min after intraventricular injection (5 microliters). The analgesic effect was calculated as the individual percent increase in the pain threshold and was compared to controls (saline treatment). The substitution of D-lysine and D-ornithine in position 2 in connection with a cyclization through ring closure of the 2 position side chain amino group to the C-terminal glycine-COOH group resulted in high analgesic potency. The substitution of D-Pro4 was without any effect in the ineffective linear derivatives and decreased the effectiveness in the highly effective cyclic derivatives. The cyclic [D-Orn2]CM-5 and the cyclic [D-Lys2]CM-5 are the CM derivatives with the highest antinociceptive activity. The cyclic [D-Orn2]CM-5 is greater than 1000 times more effective than morphine. We conclude, on the basis of studies of receptor binding and in vitro investigations, that mu receptor activity alone is not responsible for the analgesic activity. The delta receptor and possibly also the kappa receptor could modulate the nociceptive effectiveness.

Kindling and its consequences on learning in rats.

To study the learning performance of pentylenetetrazol- and amygdala-kindled Wistar rats we used the following learning tests: short-term memory was tested in the response-to-change model, brightness discrimination was tested in a Y-chamber, and two-way active avoidance learning was tested in a shuttle-box. Short-term memory was not impaired by both kindling procedures. Considering two-way active avoidance learning the performance of pentylenetetrazol (PTZ)-kindled rats was significantly diminished. This effect persists over a period of 4 weeks. However, amygdala (AMY)-kindled rats acquired this task like the controls. In brightness discrimination reaction (BDR) the learning performance of PTZ-kindled animals was not influenced. Although the acquisition of BDR was nearly identical, the 24-h retention was remarkably diminished in AMY-kindled rats. It was hypothesized that the different kindling procedures interfere in different ways and extent with neuronal circuits resulting in different functional impairments.

Influence of modified casomorphins on yawning behavior of rats.

Apomorphine-induced yawning was completely suppressed in animals treated with 5 nmol [D-Pro4]casomorphin (CM) (ICV), 10 nmol [D-Phe3]CM (ICV) or 10 nmol [D-Pip4]CM (ICV). The apomorphine-induced yawning was also decreased, by des-Tyr analogs, but only by about 50%. Physostigmine (0.15 mg/kg, IP) induced yawning. The physostigmine-induced yawning was suppressed by 5 nmol [D-Pro4]CM and 10 nmol [D-Phe3]CM. Both [des-Tyr-D-Phe3]CM and [des-Tyr-D-Pip4]CM were without effect, whereas [des-Tyr-D-Pro4]CM increased significantly the physostigmine-induced yawning. The results suggest that dopaminergic transmission can be modulated by beta-casomorphin derivatives, thus resulting in a decrease in yawning. In the case of the des-tyrosine derivatives, we can assume a dopaminergic modulation, too. An increase in serotonergic activity might be supposed for [des-Tyr-D-Pro4]CM.

Colchicine-induced lesion of rat hippocampal granular cells prevents conditioned active avoidance with perforant path stimulation as conditioned stimulus, but not conditioned emotion.

Successful acquisition of active avoidance by rats with low frequency (15 cps) stimulation of the perforant path as a conditioning stimulus is correlated with a slowly developing long-term enhancement of perforant path-granular cell synapses. After selective destruction of granular cells of the stimulated side by unilateral microinjection of 1.6 micrograms/0.2 microliter colchicine into the dentate area, field potentials could no longer be evoked by test stimuli and animals subsequently failed to acquire the conditioned active avoidance with perforant path stimulation as a CS. However, colchicine-treated animals showed the same development of conditioned emotional responses as saline controls and they could also successfully be conditioned with light and tone as the CS. These results suggest that the granular cells are necessarily involved in the conditioning pathway for the active avoidance with perforant path stimulation as the CS. Other targets of the perforant path, e.g., ipsi- and contralateral CA1 pyramidal cells and contralateral granular cells, or antidromic activation of the entorhinal cortex seem an insufficient substitute for granular cells in the pathway for this conditioned active avoidance, but would probably participate in the conditioned emotional responses. The results additionally support our hypothesis, that post-conditioning LTP in granular cell synapses contribute to the acquisition and/or the storage of a memory trace.

The analgesically effective peptide deprolorphin exhibits cross-tolerance to morphine in rats.

The potent analgesically active beta-casomorphin derivative, D-Pro4-beta-casomorphin 1-5 (= Deprolorphin), was tested for its capacity to induce tolerance. In animals tolerant to morphine, a previously analgesically effective test dose of the peptide was without any antinocipetive effect, demonstrating a cross-tolerance between morphine and deprolorphin.

Influence of postnatal hypoxia on learning and memory formation of adult rats.

Male Wistar rats were exposed to a single normobaric hypoxic treatment at the age of either 15 or 20 days. Two different kinds of hypoxia were used: 4% O2 for 6 h or 3.5% O2 for 40 min. At the age of 8 weeks the acquisition and retention of a brightness discrimination was investigated in all animals. The acquisition of all treated animals did not differ from the corresponding controls. Both hypoxic treatments at the 20th day resulted in a significant improvement of retention in the adult age, whereas only hypoxia with 3.5% O2 for 40 min at the 15th day showed a similar effect, the treatment with 4% O2 for 6 h at this time was ineffective.

Perinatal linoleate deprivation impairs learning and memory in adult rats.

Rats receiving polyunsaturated fatty acid (PUFA) deficient diets during the perinatal period showed in adult age undisturbed acquisition of a footshock motivated brightness discrimination task, but a significant impairment of retention. The same effects on retention were obtained in rats receiving the PUFA deficient diet in adulthood, when the behavioral parameters were investigated at the end of the dietary treatment.

Memory retention in old rats: improvement by orotic acid.

The effect of methylglucamine orotate (MGO) on learning and memory was investigated in 24-month-old rats using brightness discrimination in a Y-chamber and active avoidance in a shuttle box. In both learning procedures, an improvement of memory retention following 5-day MGO treatment (225 mg/kg per day) was observed. The retention of untreated old animals was significantly lower compared to 8-week-old rats. MGO treatment resulted in a significant improvement of retention in old rats, which nearly compensated for their memory deficit.

The persistence of the effect of methylglucamine orotate on the retention of a learned behavior in the rat.

The effects of methylglucamine orotate (MGO) on the retention of a brightness discrimination in rats was investigated after different intervals between treatment and acquisition, 225 mg MGO/kg were injected intraperitoneally each day over a period of 10 days. Control animals received a NaCl-solution. After drug-free intervals of 5 days or 12 days respectively, MGO-treated rats showed significantly better retention of the learned reaction than controls, whereas their training performance was not influenced. Possible interpretations of the long-lasting persistence of the orotate effect are discussed.

Postnatal orotate treatment: effects on learning and memory in adult rats.

During the postnatal period, male Wistar rats were treated with orotate, either from the 6th to 15th, 16th to 25th, or 26th to 35th day of life. Learning and memory were tested in adulthood. Rats that received orotate from the 6th to 15th day showed a better retention of a learned brightness discrimination (Y-maze) than controls. An active avoidance (pole jumping) was learned more quickly by the rats orotate-treated from the 6th to 15th day than by controls. The spontaneous locomotor activity of previously orotate-treated rats was the same as in controls. Body weight measurements revealed no differences between orotate rats and control rats. The results suggest that memory retention in adulthood can be improved by postnatal orotate treatment.

Fucose incorporation into rat hippocampus structures after acquisition of a brightness discrimination. A histoautoradiographic analysis.

Using a brigthness discrimination model in rats, the labeling of discrete hippocampus formation structures was studied after intraventricular application of [3H]-fucose. This substance was injected 5 min before training as well as 5 min, 3, 7 and 23 h after training, the pulse period lasting 120 min in all cases. A significantly training-related enhanced labeling of CA1, CA3 and CA4 cell bodies and fibres revealed that a biphasic time course occurring when radioactive fucose was applied 5 min before training and 7 h after training, whereas the increased labeling of area dentata structures was evidenced only after application of radioactive fucose 5 min before and after training. In all structures under investigation the training-related increase in labeling was more pronounced in the fibre layers than in the pyramidal and granular cell bodies.