Delta-sleep-inducing peptide and its analogs and the serotoninergic system in the development of anticonvulsive influences

Experiments on rats were carried out to study the effects of administration of delta-sleep-inducing peptide (DSIP) and its analogs (9–14) into the reticular part of the substantia nigra and ventral hippocampus on picrotoxin- and kainate-induced epileptic activity. Additionally, the uptake of [³H]tryptophan by brain structures was studied. Intranigral and intrahippocampal microinjections of peptide and its analogs were found to have anticonvulsant effects against both picrotoxin- and kainate-induced epileptic activity. Studies of the effects of DSIP and its structural analogs on the uptake of tryptophan by brain structures showed that peptides predominantly increased uptake of this amino acid. It is suggested that brain structures which modulate tryptophan uptake are largely responsible for the anticonvulsant actions of DSIP and its analogs. The results obtained here provide evidence that the serotoninergic system, is not of key importance in mediating the anticonvulsant effect of DSIP and its analogs. Translated from Rossiiski Fiziologicheskii, Zhurnal imeni I. M. Sechenova, Vol. 83, No. 8, pp. 39–45, August 1997.     

Structural-Functional Organization of Neurons in the Cerebral Cortex of Rats with Different Levels of Resistance to Emotional Stress in Conditions of Exposure to Delta Sleep-Inducing Peptide

Wistar rats with different levels of resistance to emotional stress (ES) were subjected to stress and brain sections stained with Nissl cresyl violet were used for quantitative analysis of the structural organization of neurons in layer V of the sensorimotor cortex. Some animals received delta sleep-inducing peptide (DSIP) 1 h before stress. Control ES-susceptible rats, as compared with resistant rats, had lower levels of normochromic and moderately hypochromic neurons. Normochromic neurons were not seen after stress. Rats susceptible to ES showed particularly sharp decreases in moderately hypo- and hyperchromic neurons, along with increases in the proportions of extremely hypo- and hyperchromic neurons, ghost cells, and ischemically altered cells. After administration of DSIP before stress, ischemically altered cells were not seen in any group: the level of reduction of extremely hyperchromic neurons was smaller in ES-susceptible rats than in ES-resistant rats. It is suggested that brain hypoxia plays a particular role in disorganizing the cortex in conditions of ES, while DSIP has both antistress and antihypoxic properties.     

Sleep-wave activity of a delta sleep-inducing peptide analog correlates with its penetrance of the blood-brain barrier

Delta sleep-inducing peptide (DSIP) significantly increases delta wave electrical activity in the brain of rats after intraperitoneal (i.p.) injection. Rats (n = 10) were peripherally injected with DSIP and [D-Ala4]DSIP-NH2 during the dark portion of a 12-h light/dark cycle prior to recording of epidural encephalographic (EEG) wave forms. Administration of [D-Ala4]DSIP-NH2, an analog that enters the brain after peripheral administration more readily than the parent DSIP molecule, resulted in significantly more delta waves than DSIP together with a highly significant amount of theta activity. DSIP was found to significantly increase EEG output in the delta range when compared with controls. In addition, the DSIP analog significantly decreased locomotor activity, whereas DSIP itself was without effect. These findings strongly support the controversial concepts that peripherally injected peptides can reach the brain and that DSIP compounds can increase sleep activity.     

Influence of delta-sleep inducing peptide on melatonin synthesis in the rat pineal gland

The influence of delta-sleep inducing peptide (DSIP) on rat pineal N-acetyltransferase (NAT) activity and melatonin levels was examined. Young adult rats received an injection of either saline or DSIP (either 15, 30 or 60 nmol/kg) at 20:00 h, immediately before lights out. DSIP at a dose of 15 nmol/kg significantly retarded the nighttime rise of pineal NAT activity and melatonin levels at 3 h into the dark phase. At the other two nocturnal time points (01:00 and 03:00 h) and the following morning (08:00 h) pineal NAT activity and melatonin levels were similar in all groups. The results confirmed a short-lived, dose-specific effect of DSIP on serotonin metabolism in the rat pineal gland.     

Delta sleep-inducing peptide-like material in rat brain as determined by enzyme immunoassay: Effect of sleep deprivation

The regional distribution of immunoreactive delta sleep-inducing peptide (IR-DSIP) and the effect of 24-h sleep deprivation on the IR-DSIP content were investigated in the rat brain by means of enzyme immunoassay (EIA). Most of the assayable IR-DSIP in the rat brain coeluted with authentic DSIP in the gel filtration. It is thus suggested that the present EIA method preferentially detects free DSIP and not bound DSIP. IR-DSIP proved to be more concentrated in the limbic structures, although the contents were generally at a low level. Sleep deprivation and subsequent rebound sleep had no significant effect on the brain contents of ‘free-form’ DSIP.     

Morphochemical study of hippocampus of August rats after systemic treatment with amphetamine followed by injection of delta sleep-inducing peptide

Quantitative interferometry showed that chronic amphetamine administration to August rats (2.5 mg/kg/day for 3 weeks) increased the area of neuronal cytoplasm and nuclei and content and concentrations of proteins in hippocampal CA3 neurons. These changes persisted after single injection of delta sleep-inducing peptide (60 μg/kg). The reaction of the entire neuronal population of hippocampal CA3 neurons to amphetamine is similar. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 455–457, April, 2006     

Nuclear magnetic resonance determination of flow, lactate, and phosphate metabolites during amphetamine stimulation of the rat brain

The effects of amphetamine on rat cerebral blood flow, lactate, and phosphate levels were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Proton NMR imaging was used to determine the region of brain being studied. Blood flow was estimated by detecting the washout of trifluoromethane using 19F NMR. A dose of 20 mg/kg D-amphetamine sulphate stimulated flow fourfold over control values for at least 1 h. Even though amphetamine stimulated a large increase in flow, there were no significant changes in levels of inorganic phosphate, ATP, phosphocreatine, pH or Mg2+ as determined by 31P NMR. This was true for doses of 10 and 20 mg/kg D-amphetamine sulphate. No significant changes occurred in 1H NMR detected levels of lactate with a 20 mg/kg dose of amphetamines. Lactate levels increased slightly 30 min after administration of 10 mg/kg D-amphetamine sulphate. The results call into question the central role of changes in levels of phosphates in coupling increased cortical activity to increases in metabolism.     

DSIP reduces amphetamine-induced hyperthermia in mice

The effects of delta sleep-inducing peptide (DSIP) have not been fully determined. Besides sleep-inducing activities, effects on locomotor behavior, stress-reduction, and temperature-regulation have been published. It was reported that DSIP reversed the increase in temperature of rats injected at room temperature with 15 mg d-amphetamine per kg body weight. We examined this effect in mice with 9 different concentrations of DSIP in addition to D-Ala4-DSIP and an analog, DSIP-P, phosphorylated at the serine in position 7. A reduction of the increased temperature was observed in mice but not in rats. This effect was only significant after pretreatment with 0.1 and 150 nmol DSIP/kg, but not the other doses. D-Ala4-DSIP decreased the rise in temperature between 50-150 nmol/kg, but DSIP-P showed no such effect nor was DSIP able to significantly reduce the increase of temperature induced by the larger dose of 30 mg/kg d-amphetamine. A bell-shaped dose-response curve was found for D-Ala4-DSIP; for DSIP two active dose ranges were observed. Thus, complex dose-effect relationships seem to exist for DSIP (and perhaps its analogs) in thermoregulation.     

Metabolism of individual phospholipid fractions in the rat brain during thiopental sleep and amphetamine excitation

The intensity of metabolism of the phosphate groups of all rat brain phospholipids studied was considerably reduced during sleep induced by thiopental (100 mg/kg), and distinctly increased during excitation by amphetamine (6 mg/kg). The content of diphosphoinositides rose by 39% during thiopental sleep and fell by 6% in amphetamine excitation. The content of the remaining phospholipid fractions studied was unchanged. Metabolism of the phosphoinositide fractions was more closely connected with the energy metabolism of the brain tissue than with the level of CNS functional activity.     

Effect of modafinil and amphetamine on the rat catecholaminergic neuron activity.

We have studied the effect of modafinil and amphetamine, two waking drugs, on the electrical activity of central dopaminergic and noradrenergic neurons in the rat. Modafinil (128 mg/kg, i.p.) was unable to modify the firing pattern of these neurons, while amphetamine (2 or 5 mg/kg, i.p.) consistently inhibited their activity. A pretreatment with modafinil did not change thereafter the effect of amphetamine. Contrary to amphetamine, the waking effect of modafinil does not seem to be mediated by the catecholaminergic neuron activity per se.     

Effects of chronic amphetamine treatment on the glutamate concentration in cerebrospinal fluid and brain: implications for a theory of schizophrenia

In rats, chronic (12 days) amphetamine administration (5 mg/kg, s.c.) resulted in more than 30% decrease of the glutamate content in the cerebrospinal fluid (CSF) and concomitant increase of glutamate levels in the frontal cortex, striatum and hippocampus. In contrast, chronic amphetamine had no effect on the GABA contents in these areas. The data are compatible with the interpretation that amphetamine induces an increase of dopaminergic function in these brain regions which results in an enhanced inhibition of glutamate release. It is hypothesized that diminished glutamate release accompanies the amphetamine psychosis as well as schizophrenia.     

Structural and functional organization of the cerebral cortex neurons in rats with various resistance to emotional stress following administration of delta-sleep-inducing peptide

In Wistar rats with different resistance to emotional stress (ES), subjected to stress exposure (SE), the structural organization of neurons in layer V of sensomotor cortex was studied quantitatively in brain sections stained using Nissl's cresyl violet method. One group of animals was injected with delta sleep-inducing peptide (DSIP) 1 hr before SE. In rats of control group predisposed to ES, the amount of normochromatic and moderately hypochromatic neurons was decreased as compared to the rats resistant to ES. After SE, normochromatic neurons were not demonstrated. In rats predisposed to ES, the contents of moderately hypo- and hyperchromatic neurons was found to fall dramatically with a simultaneous increase in the number of extremely hypo- and hyperchromatic neurons, ghost cells and ischemically changed cells. After DSIP infusion before SE, ischemically damaged cells were not found in any group, while the degree of shrinkage of extremely hyperchromatic neurons was lower in rats predisposed to ES as compared to rats resistant to ES. It is suggested that brain hypoxia plays an important role in cortex disorganization during ES, whereas DSIP, possesses both antistress and antihypoxic effects.     

The regional distribution of d-amphetamine and local glucose utilization in rat brain during continuous amphetamine administration

Summary The distribution of radioactivity following administration of either [³H]d-amphetamine or [³H]2-deoxy-d-glucose was examined by scintillation counting of 22 microdissected brain regions from rats pretreated with either acute or continuous amphetamine, or continuously administered labeled d-amphetamine. Animals continuously administered drug were sacrificed in behaviorally distinct stages of the continuous amphetamine syndrome, a potential animal model of amphetamine psychosis.Both isotopes were heterogeneously distributed within brain, and their distributions were differentially affected by acute or continuous amphetamine regimens. While the distribution of either isotope in naive rats was characterized by greatest concentrations of counts in rostral rather than caudal regions, and grey-matter rather than white-matter structures, continuous amphetamine administration resulted in progressively increased retention of amphetamine by mesolimbic but not nigrostriatal brain regions; this was accompanied by locally enhanced levels of glucose utilization. This effect was predominantly localized in the nucleus accumbens, which exhibited the greatest retention of amphetamine and greatest relative increase in glucose utilization of any region studied during that stage of the continuous amphetamine syndrome thought to best model amphetamine psychosis. Alterations in amphetamine distribution and local levels of neural activity may reflect a change in the principal locus of control of amphetamine effects within brain as animals progress through the stages of the continuous amphetamine syndrome.Supported by U.S.P.H.S. grant DA 02312     

Expression of the c-fos Gene during Emotional Stress in Rats: The Clocking Effect of Delta Sleep-Inducing Peptide

Emotional stress induced more marked increases in the expression of the c-fos gene in limbo-reticular structures of the brain in rats prognostically predisposed to emotional stress. I.p. doses of delta sleep-inducing peptide (DSIP) (60 nmol/kg) weakened the stress-induced expression of the c-fos gene. This effect was more apparent in animals predisposed to emotional stress, in which preliminary injections decreased stress-induced c-fos expression in the paraventricular hypothalamus and the medial and lateral parts of the septum. The decreased expression of the early gene c-fos in emotional stress after preliminary dosage with DSIP may reflect the leading mechanism of the anti-stress action of this peptide.     

In vivo modulation of ventral tegmental area dopamine and glutamate efflux by local GABA(B) receptors is altered after repeated amphetamine treatment

The activity of dopamine neurons in the ventral tegmental area is modulated by excitatory (glutamatergic) and inhibitory (GABAergic) afferents. GABA, released by intrinsic neurons and by projection neurons originating in the nucleus accumbens and other regions, inhibits dopamine neurons via activation of GABA(A) and GABA(B) receptor subtypes. Using in vivo microdialysis in freely moving rats, we investigated the role of ventral tegmental area GABA(B) receptors in modulating levels of dopamine and glutamate within the ventral tegmental area, both in naive rats and in rats treated repeatedly with saline or amphetamine (5 mg/kg i.p., for 5 days). In naive rats, administration of a potent and selective GABA(B) receptor antagonist (CGP 55845A) into the ventral tegmental area elicited a concentration-dependent increase in dopamine levels, but did not alter glutamate levels. In rats tested 3 days after discontinuing repeated amphetamine administration, 50 microM CGP 55845A increased dopamine levels to a greater extent than in saline controls. This difference was no longer present in rats tested 10-14 days after discontinuing repeated amphetamine injections. CGP 55845A (50 microM) had no effect on glutamate levels in the ventral tegmental area of saline-treated rats. However, it produced a robust increase in glutamate levels in rats tested 3 days, but not 10-14 days, after discontinuing repeated amphetamine injections.These results suggest that somatodendritic dopamine release is normally under strong tonic inhibitory control by GABA(B) receptors. Repeated amphetamine administration enhances GABA(B) receptor transmission in the ventral tegmental area during the early withdrawal period, increasing inhibitory tone on both dopamine and glutamate levels. This is the first demonstration, in an intact animal, that drugs of abuse alter GABA(B) receptor transmission in the ventral tegmental area.     

NO-ergic rat brain commissural neurons in the norm and during opiate administration

Using histochemical reaction demonstrating NADPH-diaphorase (NADPH-d), the dynamics of NO synthesis was studied in the rat brain raphe nuclei following intravenous injection of morphine hydrochloride. In normal conditions NADPH-d activity was demonstrated in neurons of all raphe nuclei. Acute and chronic administration of morphine in different doses (0.5 mg/kg and 5 mg/kg) was found to inhibit NO-ergic activity of the major part of raphe nuclei neurons. The depression of NADPH-d activity was unequal in different nuclei. The NO-ergic changes are caused by an activation of opiate receptors, as they depend on morphine dose, while the application of opiate antagonist naloxone restores the NO-ergic function of raphe neurons. Formation of tolerance to opiate analgetic effect is accompanied by a significant, though short-lasting increase of NO synthesis activity. It is suggested that the changes in NO-ergic function of raphe neurons may influence brain serotonin balance after opiate administration.     

Central noradrenaline depletion attenuates amphetamine-induced locomotor behavior

Male rats were given 6-hydroxydopamine-induced lesions of the locus coeruleus (LC) or the dorsal noradrenergic bundle (DNAB), prior to the measurement of locomotor and rearing activity induced by D-amphetamine. The increased locomotor activity induced by D-amphetamine (1.8 mg/kg) was significantly attenuated by both the LC and the DNAB lesions. The stimulatory effect of the 7.2 mg/kg dose of amphetamine was attenuated by the LC lesion, whereas the DNAB lesion potentiated this effect. The LC lesion also attenuated rearing induced by the 7.2 mg/kg dose of amphetamine. These results suggest some involvement of central noradrenergic neurons in the activity induced by amphetamine in the rat.     

Effect of delta-sleep inducing peptide on electrical instability of the heart in emotional stress

The effect of the delta-sleep inducing peptide (DSIP) on disorders of the cardiac rhythm in emotional stress was studied in experiments on rabbits. DSIP (60 nmol/kg) diminished or arrested ventricular extrasystole occurring in experimental emotional stress. It was shown that the antiarrhythmic effect of DSIP is most manifest if it is injected just before exposure to the stress factors. DSIP injection induces increase of the thresholds of the occurrence of ventricular fibrillation and its precursors in intact animals. This can explain the high preventive effect of DSIP in stress. The acquired data on the antiarrhythmic effect of DSIP should be taken into consideration in elaborating preventive recommendations aimed at raising the organism's stability to stress factors.     

Two-way shuttlebox avoidance conditioning and brain NADH in rats

The effects of amphetamine (1.5 mg/kg) and caffeine (120 mg/kg, 75 mg/kg) on shuttle behavior and on the concentrations of reduced nicotinamide adenine dinucleotide (NADH) of the brain were studied in 56 rats from the perspective of regional brain metabolism. Amphetamine potentiated the shuttle behavior including avoidance responses and response speed, while it increased NADH concentrations in the hippocampus. The low dose of caffeine increased avoidance responses. The high dose of caffeine inhibited avoidance responses, but it had no effect on NADH concentrations in discrete brain regions.     

Changes in noradrenergic terminal excitability induced by amphetamine and their relation to impulse traffic

The effects of amphetamine upon the terminal excitability of noradrenergic neurons of the nucleus locus coeruleus were studied in urethane anesthetized rats. Terminal excitability was measured by determining the stimulus currents necessary to evoke antidromic responses in locus coeruleus neurons from terminals in the frontal cortex. In most cases, terminal excitability was decreased following local infusion of amphetamine into the frontal cortex, while intravenous administration of the drug tended to increase terminal excitability. The decreased terminal excitability induced by local infusion of amphetamine appeared to be due to activation of alpha-adrenergic receptors located on the terminals of locus coeruleus neurons, since this effect mimics that of clonidine, a direct acting alpha-adrenergic agonist, and since the effect was abolished by pretreatment with alpha-methyl-p-tyrosine which disrupts the catecholamine liberating properties of amphetamine. Phentolamine, a direct acting alpha-adrenergic receptor antagonist was also found to block or reverse the effect of amphetamine. The changes in terminal excitability following intravenous injection of amphetamine appeared to be related to changes in the spontaneous activity of locus coeruleus neurons. A large decrease in spontaneous activity following intravenous administration of amphetamine was associated with increased terminal excitability, whereas when smaller changes in spontaneous activity occurred, terminal excitability was found to be decreased. These results are discussed with respect to the pharmacological properties of catecholaminergic neurons and the mechanisms of action of amphetamine.