Effects of Neonatal Treatments on Pain and Audiogenic Sensitivity and Brain Content of Monoamines in Adult Rats

In adult Wistar, KM, and Wag/Rij rats, the threshold of pain sensitivity (tail-flick test) and audiogenic sensitivity were estimated after neonatal administration of Semax (analog of ACTG_4–10 fragment) or after placebo (administration of saline for the control of the effect of neonatal pain stimulation). These neonatal treatments did no affect the rates of sensomotor development at an early age (Fox tests), i.e., did not affect directly the physiological activity of rat pups at the age of up to 21 days. In all control rats injected with saline (pain stimulation), the latencies of audiogenic fits increased reliably, while their degree decreased. Administration of Semax raised these parameters to the level of those in intact animals, i.e., increased the sensitivity to sound. Neonatal administration (per os) of caffeine to KM rats increased reliably the latency of audiogenic fits. The thresholds of pain sensitivity in the rats of all strains were significantly lower saline injected animals than in the intact control, just as the level of dopamine in the hippocampus of KM rats. These data are interpreted as an evidence of changes in the development of some brain systems in response to neonatal treatments.     

The influence of neonatal ketamine injection on pain sensitivity and audiogenic seizures in adult rats

The remote effects of neonatal (on the 3d-to-9th postnatal days) ketamine injections (10 and 50 mg/kg in 20 microliters of distilled water, s.c.) were analyzed in adult Wistar, WAG/Rij, and KM (a strain with high audiogenic sensitivity) rats. Both ketamine and water injections increased pain sensitivity in adult rats. Neonatally injected water increased the mean score of seizures in Wistar and WAG/Rij, whereas ketamine water solution injected in the dose of 50 mg/kg did not change the seizure intensity (as compared to the intact control). Consequently, ketamine significantly reduced the mean score of the audiogenic seizure fit without change in its latency. In highly sensitive KM rats the neonatally injected ketamine (50 mg/kg) significantly shortened the mean latency of the fit onset, and fit stages developed faster. Thus, the neonatal ketamine injection increased the audiogenic seizure susceptibility of brain structures in KM rats.     

Audiogenic kindling changes the subunit composition of BK-channels in dentate gyrus of Krushinskii-Molodkina rats

Subunit composition of voltage- and Ca²⁺-sensitive high-conductance K⁺ channels (BK channels) in dentate gyrus (DG) of Krushinskii-Molodkina (KM) rats, genetically prone to audiogenic seizures, was compared with that of normal Wistar rats, resistant to sound effects. Additionally, long-lasting changes in protein expression of α- and β4-subunits in DG of KM rats after audiogenic kindling (model of temporal lobe epilepsy) was investigated. Western blot analysis revealed no differences between the levels of the pore-forming α-subunit expression in DG of KM and Wistar rats. In contrast, the level of brain-specific auxiliary β4-subunit in DG of KM rats was increased twofold in comparison to that in Wistar rats. It is likely that the observed changes in the BK channel α/β4 subunits ratio can prevent the development of excessive neuronal exitability in DG of KM rats. The results obtained on the model of audiogenic kindling (20 convulsion fits) confirmed this assumption. Thus, α-subunit expression levels in DG of KM rats on day 3 and 14 after the last seizure were increased 2.5 times in comparison with intact KM rats. The expression level of β4 in DG of KM rats 3 days after kindling was reduced to 30% of the control level. On day 14 after finishing audiogenic kindling, a further reduction of β4 protein expression level occurred. We suggest that the changes in the subunit composition of BK channels in DG following chronic seizures can alter functional properties of DG as a physiological filter, which normally prevents the propagation of epileptiform activity in the hippocampus.     

Electroencephalographic analysis of manifestations of latent forms of paroxysmal syndrome in the wakefulness-sleep cycle in rats

An electroencephalographic study of the brain activity in the wakefulness-sleep cycle was carried out on rats of Krushinskii-Molodkina line (KM) with hereditary predisposition to audiogenic convulsions and on Wistar rats that were insensitive to the convulsiogenic sound effect, but with epileptiform manifestations appearing on the background of cadmium intoxication and administration of kainic acid into the caudate nucleus head. There were revealed several EEG patterns whose presence was an indicator of formation of disorders of the CNS activity of the paroxysmal character in the animals. It has been established that in the phase of the rat rapid-wave sleep, a high representation of episodes with predominance of a-diapason EEG oscillations can be considered a specific non-paroxysmal abnormality due to the presence of convulsive syndrome in these animals. It was shown the long steady decrease of sensitivity of KM rats to the convulsiogenic sound effect, which appeared after multiple audiogenic generalized tonicoclonic convulsive attacks, correlated with a decrease of the degree of theta-diapason oscillations and with an increase of representation of alpha-diapason waves on EEG in the state of the animal quiet consciousness. A role of disintegration in activity of the ascending activating brain systems in the animal and human paroxysmal syndromes is discussed.     

Morphofunctional features of microtubule ultrastructure and MAP2 protein phosphorylation in hippocampal neurons of rats with predisposition to audiogenic epilepsy

It has been shown that modification of microtubule (MT) ultrastructure are accompanied by functional changes in microtubule-associated protein MAP2 in the hippocampus of Krushinsky--Molodkina rats (KM), which are prone to autogenic seizures. The morphogenetic analysis revealed that contrary to Wistar rats, which are insensitive to sound stimulation, in KM the middle length of microtubule fragments in the apical dendrites of pyramidal neurons in CA3 hippocampal area was reduced. Using immunoblot and autoradiography methods, we found that the level of MAP2 and the rate of its cAMP = and Ca(2+)-calmodulin-dependent phosphorylation were increased in hippocampus of KM, in comparison with Wistar rats. Daily repeated sound stimulation for 20 days (audiogenic kindling) induced a further decrease in length of MT fragments, and an increase of their density in the proximal part of apical dendrites of KM. Moreover, audiogenic kindling induced additional increase in MAP2 phosphorylation state, but did not change the level of MAP2 in KM hippocampus. We suppose that the obtained alteration of MAP2 phosphorylation state exerted influence on kinetic parameters of microtubule assembly, serving as part of genetically determined predisposition of KM to audiogenic epilepsy.     

Electroencephalographic analysis of manifestations of hidden forms of paroxysmal syndrome in the wakefulness-sleep cycle in rats

An electroencephalographic study of the brain activity in the wakefulness-sleep cycle was carried out on rats of Krushinskii-Molodkina line (KM) with hereditary predisposition to audiogenic convulsions and on Wistar rats that were insensitive to the convulsiogenic sound effect, but with epileptiform manifestations appearing on the background of cadmium intoxication and administration of kainic acid into the caudate nucleus head. There were revealed several EEG patterns whose presence was an indicator of formation of disorders of the CNS activity of the paroxysmal character in the animals. It has been established that in the phase of the rat rapid-wave sleep, a high representation of episodes with predominance of α-diapason EEG oscillations can be considered a specific non-paroxysmal abnormality due to the presence of convulsive syndrome in these animals. There was shown a long steady decrease of sensitivity of KM rats to the convulsiogenic sound effect, which appeared after multiple audiogenic generalized tonicoclonic convulsive attacks, correlated with a decrease of the degree of ?-diapason oscillations and with an increase of representation of α-diapason waves on EEG in the state of the animal quiet wakefulness. The role of disintegration in activity of the ascending activating brain systems in the animal and human paroxysmal syndromes is discussed.     

Remote effects of early postnatal pituitary hormone melatonin injection on audiogenic seizures in Krushinsky-Molodkina rats

Rats Krushinsky-Molodkina inbred strain (KM) genetically prone to audiogenic seizures were injected with pineal hormone melatonin (50 mg/kg, s.c.) within the period 7th to the 14th posnatal days (PND). The remote effects of this injection adult KM rats consisted in a decrease in the latency and increase in severity of myoclonic seizures produced by audiogenic kindling (20 sound stimuli, 100 dB and 12-15 kHz). As compared to the control, in the cortex and hippocampus of rats of melatonin group, we also found a significant reduction of both total and functional activity of Ca2+/calmodulin-dependent protein kinase II (CAMK II) after audiogenic kindling. On the contrary, melatonin administration within the 1st to 7th PND and the 14th to the 21st PND resulted in a decrease in seizure activity. In the first case, both the total (cortical) and functional (hippocampal) CAMK II activities in melatonin-injected rats were increased as compared to control, whereas in the second case, only a slight increase in Ca2+-independent CAMK II activity in the hippocampus of melatonin-injected rats was observed. Probably, the melatonin administration in the period of early postnatal development changes the features of expression and/or regulation of CAMK II activity, and this could be one of the mechanisms of audiogenic seizure modulation in KM rats.     

The interstrain differences in the effects of D-amphetamine and raclopride on dorsal striatum dopaminergic system in KM and Wistar rats (microdialysis study)

The levels of dopamine (DA) was determined by intracerebral microdialysis in vivo in KM rats selected for high audiogenic epilepsy, and in Wistar rats selected for nonsusceptibility to loud sound. The basal level of dopamine was 25% higher in the KM rats (P < 0.05). A single amphetamine injection (1 mg/kg body weight, intraperitoneously) caused a significant increase in the DA basal level up to 250-260% in animals of both genotypes. However, in Wistar rats, the level of DA reached maximum as soon as 20 min after amphetamine administration, whereas in KM rats, this happened only after 120 min. After a single injection of the antagonist of D2 and D3 dopamine receptors raclopride (1.2 mg/kg of body weight, intraperitoneously), an increase in the level of DA was similar in amplitude in rats of both genotypes (up to about 210%); however, this occurred 20-30 and 100 min after raclopride administration to Wistar and KM rats, respectively. This evidence suggests that the genetic defect of KM rats, namely, the high level of audiogenic epilepsy, is caused by abnormalities of the neuromediator brain systems and presumably accompanied by the regulatory gene dysfunction.     

The Interstrain Differences in the Effects of D-Amphetamine and Raclopride on Dorsal Striatum Dopaminergic System in KM and Wistar Rats (Microdialysis Study)

The levels of dopamine (DA) was determined by intracerebral microdialysis in vivo in KM rats selected for high audiogenic epilepsy, and in Wistar rats selected for nonsusceptibility to loud sound. The basal level of dopamine was 25% higher in the KM rats (P < 0.05). A single amphetamine injection (1 mg/kg body weight, intraperitoneously) caused a significant increase in the DA basal level up to 250-260% in animals of both genotypes. However, in Wistar rats, the level of DA reached maximum as soon as 20 min after amphetamine administration, whereas in KM rats, this happened only after 120 min. After a single injection of the D2/D3 dopamine receptor antagonist raclopride (1.2 mg/kg of body weight, intraperitoneously), an increase in the level of DA was similar in amplitude in rats of both genotypes (up to about 210%); however, this occurred 20-30 and 100 min after raclopride administration to Wistar and KM rats, respectively. This evidence suggests that the genetic defect of KM rats, namely, the high level of audiogenic epilepsy, is caused by abnormalities of the neurotransmitter brain systems and presumably accompanied by the regulatory gene dysfunction.     

A decrease in thymus-mediated immune responses as a result of treatment of neonatal rats with glutamate

We investigated whether administration of monosodium l-glutamate (MSG) to neonatal rats would disrupt immune responses in intact and orchidectomized adult male rats. Neonatal male rats were treated with saline or MSG which causes severe endocrine abnormalities. Half of each group of animals were orchidectomized as adults and killed one week later along with intact rats. MSG treatment resulted in suppressed serum LH levels in intact rats. Thymus weight and spleen cellularity in intact animals were not affected by MSG treatment, but thymus weight increased within one week after orchidectomy in both saline- and MSG-treated groups. In intact rats, lymphocyte stimulation by the T cell specific mitogens (concanavalin A or phytohemagglutinin) or the B cell specific mitogen (lipopolysaccharide) was unaffected by prior treatment with MSG. However, MSG treatment blocked the decrease attributable to orchidectomy in concanavalin A and phytohemagglutinin stimulation of lymphocyte blastogenesis. The results suggest that administration of MSG to neonatal male rats can alter some immune responses in the adult animal.     

Energetic,oxidative and ionic exchange in rat brain and liver mitochondria at experimental audiogenic epilepsy (Krushinsky–Molodkina model)

The role of brain and liver mitochondria at epileptic seizure was studied on Krushinsky-Molodkina (KM) rats which respond to sound with an intensive epileptic seizure (audiogenic epilepsy). We didn't find significant changes in respiration rats of brain and liver mitochondria of KM and control rats; however the efficiency of АТР synthesis in the KM rat mitochondria was 10% lower. In rats with audiogenic epilepsy the concentration of oxidative stress marker malondialdehyde in mitochondria of the brain (but not liver) was 2-fold higher than that in the control rats. The rate of H₂O₂ generation in brain mitochondria of КМ rats was twofold higher than in the control animals when using NAD-dependent substrates. This difference was less pronounced in liver mitochondria. In KM rats, the activity of mitochondrial ATP-dependent potassium channel was lower than in liver mitochondria of control rats. The comparative study of the mitochondria ability to retain calcium ions revealed that in the case of using the complex I and complex II substrates, permeability transition pore is easier to trigger in brain and liver mitochondria of KM and КМs rats than in the control ones. The role of the changes in the energetic, oxidative, and ionic exchange in the mechanism of audiogenic epilepsy generation in rats and the possible correction of the epilepsy seizures are discussed.     

Effects of ionotropic glutamate receptor channel blockers on the development of audiogenic seizures in Krushinski-Molodkina rats

The action of noncompetitive blockers of glutamate receptors has been investigated on Krushinski-Molodkina rats genetically-prone to audiogenic seizures. The selective blockers of NMDA receptor channels, memantine and IEM-1921, and their dicationic homologues, IEM-1925 and IEM-1754, capable of blocking in varying degrees both NMDA and Ca-permeable AMPA receptor channels, were studied. The drugs were injected intramuscularly to rats with the different time intervals (30 min, 1, 2 or 3 hours) before sound signal. The effects of the drugs on latent period of initial locomotor activity provoked by audio stimulation (8 kHz sine-wave tone, 90 dB volume), the appearance of clonic convulsions of different intensities, and, finally, tonic convulsions with limb and tail extension were evaluated. Within 30 min after injection IEM-1921 at a dose of 5 mg/kg, 33% of rats manifested a complete absence of convulsive reactions to sound, and in 59% of rats audiogenic seizures occured only in the form of motor excitation without a generalized clonic-tonic convulsions. Memantine at a dose of 5 mg/kg did not cause a complete blockade of seizures, but after 1 h of injection in 50% of the rats and after 2 h in 70% of rats a weakening of the audiogenic seizures to the level of motor excitation only was observed. After 3 hrs after administration of blockers its anticonvulsive action weakened significantly (p < 0.01). Dicationic blockers that block both NMDA and AMPA/kainate receptors, IEM-1925 (in doses of 0.001-20.0 mg/kg) and IEM-1754 (0.025-50.0 mg/kg), did not affect audiogenic clonic-tonic convulsive reactions. The involvement of activation of NMDA and calcium permeable AMPA/kainate receptors in the pathogenesis of audiogenic seizures is discussed.     

Laboratory rat selection for the trait "the absence of audiogenic seizure proneness"

The hybrids between Krushinsky-Molodkina (KM) inbred strain, selected for high predisposition to audiogenic epilepsy (AE), and Wistar rats non-prone to audiogenic seizure were the initial population for selection. Rats were selected for the trait "the absence of audiogenic seizure proneness". The creation of such strain in which the significant proportion of animals develop no AE in response to sound and share partly the genetic background of the KM strain is very important for the correct use of RV strain as the laboratory model of seizure states. As alleles which determine the AE proneness are recessive the selection for the "opposite" trait proceeds necessarily slow.     

Laboratory rat selection for the trait “the absence of audiogenic seizure proneness”

The hybrids between Krushinsky-Molodkina (KM) inbred strain, selected for high predisposition to audiogenic epilepsy (AE), and Wistar rats non-prone to audiogenic seizure were the initial population for selection. Rats were selected for the trait ??the absence of audiogenic seizure proneness??. The creation of such strain in which the significant proportion of animals develop no AE in response to sound and share partly the genetic background of the KM strain is very important for the correct use of KM strain as the laboratory model of seizure states. As alleles which determine the AE proneness are recessive the selection for the ??opposite?? trait proceeds necessarily slow.     

Anticonvulsive properties of peptide ACTH4-7 pro-gly-pro detected in amygdaloid kindling and audiogenic epilepsy in rats

Effects of the ACTH4-7 pro-gly-pro, calcium valproate ("Germed", DDR) and nembutal on kindling preparation and audiogenic epilepsy were investigated. Development of after-discharges in response to repeated amygdaloid electrical stimulation was assessed in normal rats and in rats susceptible to audiogenic epilepsy (KM line of rats). ACTH4-7 pro-gly-pro had an anticonvulsant profile. ACTH4-7 pro-gly-pro decreased seizure threshold in the audiogenic epilepsy test, but did not prevent the motor convulsions.     

Developmental changes in audiogenic epilepsy and myoclonus in KM rats

The reaction to a single sound stimulation (a bell) applied daily and the time of the first appearance of myoclonus were recorded in Krushinsky-Molodkina (KM) rats of different ages (30, 60, and 150-200 days). The audiogenic seizures in adult animals were of short latency and always resulted in tonic convulsions, which were stable in their patten. In contrast, in young animals (in particular, in 30-day-old rats), the seizures were less pronounced and unstable. The daily sound stimulation produced a gradual development of myoclonic seizures in young and adult rats. In old rats, the stable myoclonus appeared on the 17th day of stimulation, and in 30-day-old animals the myoclonus was recorded on the 26th day. In 60-day-old rats the myoclonus appeared for the first time on the 20th day of stimulation. It is suggested that these age differences can be explained by the btain immaturity (in particular, of neurotransmitter system) in young animals.     

The remote effects of neonatal injections of caffeine and piracetam on audiogenic seizure susceptibility in mice of three genotypes

Neonatal DBA/2J, 101/HY and CBA/Lac/Sto mice (2-7-day-old) were subcutaneously injected with caffeine (200 mg/kg), piracetam (50 mg/kg) or distilled water. At the age of 1 month, they were tested for audiogenic seizure susceptibility (SS). The neonatal injections changed SS in 1-month-old mice in a genotype-dependent manner. Distilled water (control of neonatal pain stimulation) slightly reduced the audiogenic fit severity (arbitrary scores) the effect being most distinct in DBA/2J, less strong in 101/HY strain and absent in CBA. Caffeine neonatal injections induced slight changes in DBA/2J, no changes in CBA and increased SS in 101/HY mice. Piracetam reduced fit intensity in DBA/2J mice but increased it in CBA and, especially, in 101/HY strain. Genotype-dependent differences in physiological mechanisms of audiogenic seizures may be responsible for different remote effects of early treatment.     

Effects of total sleep deprivation in rats with hereditary predisposition to audiogenic convulsions

In rats of the Krushinskii-Molodkina (KM) line with hereditary predisposition to audiogenic convulsions there were studied effects of total sleep deprivation for 3, 6, and 9 h by a light arousal or a slow rotation in a roller on spectral EEG characteristics in the wakefulness-sleep cycle, organization of the cycle, and intensity of convulsive symptoms at the recovery period. The data are presented on dynamics of recovery of the cycle structure for 12 h of postdeprivation period. It has been established that during and after the total sleep deprivations of any duration no paroxysmal discharges appear in EEG of hippocampus, caudate nucleus, medial central thalamic nucleus, somatosensory, visual, and auditory cerebral cortex in any of states of the wakefulness-sleep cycle. These deprivations were also shown to have no effect on the latent period value and parameters of generalized tonic-clonal audiogenic convulsions. At the same time, after 6 and 9 h of the total sleep deprivations in a slowly rotating roller there was revealed in some animals a change of the type of response to the sound stimulus. Such decrease of reaction of rats to audiogenic stimuli seems to be due to alertness of the animals. It is stated that in the KM rats, with the hidden convulsive syndrome, we failed to activate epileptiform manifestations by the used types and ways of the total sleep deprivations.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 82–88.Original Russian Text Copyright © 2005 by Vataev, Oganesyan.     

3H-1-glutamate binding with the synaptic membranes isolated from the cerebral cortex and hippocampus of Krushinski?-Molodkina strain rats

Specific binding of 3H-L-glutamate to synaptic membranes isolated from the cerebral cortex and hippocamp of Wistar and Krushinsky-Molodkina (KM) rats examined both in a quiet awake state and after audiogenic seizures was compared. The dissociation constant (KD) values and binding capacity (Bmax) for KM rats did not differ significantly from the corresponding parameters of binding determined for Wistar rats (KD--89.8 +/- 18.1 and 102.6 +/- 12.5 nm, Bmax--1.23 +/- +/- 0.08 and 1.30 +/- 0.15 pmol/mg for the cortex and hippocamp, respectively). After audiogenic seizures the binding capacity of the hippocamp of KM rats was reduced by 30%. It is suggested that hippocampal glutamate receptors of KM rats are involved in the mechanism of convulsive activity formation.     

The effect of neonatal administration of monosodium glutamate on behavior and blood corticosterone level

DBA/2 male mice were treated with monosodium glutamate (MSG) in a dose of 4 mg/g on 1, 3, 5, 7, 9 days after birth. Saline treated and intact males were used as control groups. MSG treated males displayed decreased number of crossed squares, rearings, entries in the centre and time in the centre of open field in comparison with saline-treated but not intact animals. Time in the light compartment of the light-dark box was increased in MSG-treated mice versus both saline treated and intact animals. MSG administration reduced acoustic startle response but did not affect the magnitude of prepulse inhibition of the startle reflex. Sexual motivation in male mice was reduced by MSG, the same trend was observed after saline treatment. MSG administration increased corticosterone basal level 4-fold while saline treatment did not affect it. These data suggest that neonatal administration of MSG decreases locomotion, exploratory activity, anxiety in male mice, while corticosterone level is increased. Saline treatment increases these parameters (except sexual motivation), and this augmentation is not connected to changes in corticosterone basal level.