The role of neurotrophins in the maintenance of the spinal cord motor neurons and the dorsal root ganglia proprioceptive sensory neurons.

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non-muscle- and a few muscle-innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double-mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT-4/5, TrkB and TrkC, and not NT-3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT-4/5 and TrkC, and not NT-3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT-3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).

Antiepileptic activity of delta sleep-inducing peptide and its analogue in metaphit-provoked seizures in rats.

PROBLEM: Previous studies have shown that humoral, endogenous and somnogenic, delta sleep-inducing peptide (DSIP) has influence on insomnia, pain, adaptation to stress, epilepsy, etc. We investigated the potential of DSIP and its analogue DSIP-12 (a nonapeptide with alanine in position 2 of DSIP molecule substituted by beta-alanine) to antagonize metaphit (1-[1(3-isothiocyanatophenyl)-cyclohexyl]piperidine) induced generalized, reflex audiogenic seizures in adult male Wistar albino rats. METHODS: The rats divided in four groups received (i.p.): saline; metaphit; metaphit+DSIP; and metaphit+DSIP-12, respectively. Metaphit-treated animals displaying seizure in eight previous tests received DSIP or DSIP-12 and afterwards audiogenic stimuli were applied at hourly intervals for the next 30 h. The animals were exposed to sound stimulation 60 min after metaphit administration and further on at hourly intervals. Incidence and severity of seizures were behaviorally analyzed. Selected EEGs and power spectra were recorded and analyzed. RESULTS AND CONCLUSIONS: Metaphit led to hypersynchronous epileptiform activity (polyspikes and spike-wave complexes) and increased power spectra 0.5-30 h after the treatment. Severity of metaphit seizures increased with time to reach the peak 7-12 h after injection. DSIP and DSIP-12 significantly (*P<0.05 and **P<0.01) increased in delta and theta frequency bands and decreased the incidence, mean seizure grade and duration of metaphit convulsions. The results suggest that DSIP and DSIP-12 may be considered as potential antiepileptics in the animal model, DSIP-12 being more efficient than DSIP.

Ontogenetic study of metaphit-induced audiogenic seizures in rats.

Ontogenetic differences in susceptibility to metaphit (1-(1-(3-isothiocyanatophenyl)cyclohexyl)-piperidine)-induced audiogenic seizures were examined in young, developing (ages: 12, 18, and 25 days) and adult (90 days old) Wistar albino rats. Metaphit was injected in a dose of 10 mg/kg i.p. and animals were subjected to intense audio stimulation (100 +/- 3 dB, 60 s) at hourly intervals after administration. Audiogenic seizures (AGS) were scored according to a four point descriptive rating scale (0-3). AGS were elicited in all age groups; they were induced for 12, 15, 15, and 30 h in 12-, 18-, 25-day-old, and adult rats, respectively. Younger animals reached a peak incidence and severity of seizures before adult rats. Twenty-five-day-old rats showed greatest incidence and severity of seizures, and shortest latency. Twelve-day-old animals had longest latencies. Besides audiogenic seizures, we observed convulsions induced by metaphit only in the form of running episodes, forelimb clonus, clonic convulsions, and rearing. Results suggest that young rats develop metaphit-induced sound seizures more rapidly, but that adults have longer period of seizure susceptibility. Different susceptibility to seizures is probably due to changes in excitatory and inhibitory pathways, while maturation of blood-brain barrier is less probable, since metaphit has a lipophilic nature.

Delta sleep-inducing peptide and its tetrapeptide analogue alleviate severity of metaphit seizures.

The effects of delta sleep-inducing peptide (DSIP) and its tetrapeptide analogue, DSIP(1-4), on metaphit-induced audiogenic seizures were studied. Five groups of adult male Wistar rats were intraperitoneally treated with (1) saline, (2) metaphit, (3) DSIP, (4) metaphit+DSIP and (5) metaphit+DSIP(1-4). To examine blocking effects of DSIP and its analogue on fully developed metaphit seizures, the last two groups were injected after the eight audiogenic testing. The rats were stimulated using electric bell (on the top of the cage, generating 100+/-3 dB and frequency 5-8 kHz, for 60 s) 1 h after metaphit and afterwards at hourly intervals during the experiment. For EEG recordings and power spectra, three gold-plated screws were implanted into the skull. In metaphit-treated animals, EEGs appeared as polyspikes and spike-wave complexes while the power spectra were increasing for 30-h period. The incidence and severity of metaphit-induced audiogenic seizures reached peak value 7-12 h after the injection. Both DSIP and DSIP(1-4) significantly increased power spectra of delta waves and decreased incidence of seizures, mean seizure grade and tonic component of metaphit-induced convulsions. Taken together, these results suggest that DSIP and its analogue DSIP(1-4) should be considered as potential antiepileptics.

[Behavioral and electroencephalographic effects of delta sleep inducing peptide and its analogue on metaphit-induced audiogenic seizures in rats]. / Ponasanje i elektroencefalografska dejstva delta peptida i njegovog analoga u zvucnoj epilepsiji izazvanoj metafitom kod pacova.

INTRODUCTION: Delta sleep inducing peptide (DSIP) is well known natural somnogenic peptide that has many other physiological functions. DSIP analogues representing hepta- and octapeptides (also known as long) as well as tetrapeptide (termed short, used in our experiments) were synthesized with a view to evaluate the peptide specificity in sleep. The effects of DSIP and its analogue DSIP1-4 on metaphit 1-[1(3-isothiocyanatophenylciclohexyl)-piperidine] induced audiogenic seizures were evaluated in rats. METHODS: Male Wistar albino rats were divided into 4 groups: 1. Saline; 2. Metaphit; 3. Metaphit + DSIP, and 4. Metaphit + DSIP1-4. To examine the blocking effects of DSIP and its analogue on fully developed metaphit seizures, the last two groups were injected after the 8th audiogenic testing. Animals were injected with metaphit (10 mg/kg) intraperitoneally (i.p.) and exposed to sound stimulation (100 +/- 3 dB, 60 s) at hourly intervals. The incidence and severity (running, clonus and tonus) of seizures were analyzed. For electroencephalographic (EEG) recordings, three gold-plated electrodes were used. Convulsive behavior was assessed by incidence of motor seizure and by seizure severity grade, determined by descriptive rating scale ranging from 0 to 3: 0--no response, 1--wild running only; 2--wild running followed by clonic seizures of all four limbs with body rollover; 3--wild running progressing to generalized clonic convulsions followed by tonic extension of fore- and hind legs and tail. Sound onset, seizure events, and sound offset, along with the animal's behavior (convulsive or other) were characterized with EEG changes. RESULTS: In most animals, the administration of metaphit resulted in electroencephalographic abnormalities, elicited epileptiform activity in the form of spikes, polyspikes and spike-wave complexes. Maximum incidence and severity of metaphit convulsions occurred 8 h after the injection (9/12, 75%), then abated gradually and disappeared 30 h later. Both DSIP and DSIP1-4 significantly increased the power spectra of d waves and decreased the incidence of seizures, mean seizure grade and tonic component of metaphit-induced convulsions. DISCUSSION: Metaphit has been shown to induce audiogenic seizures after systemic and intracerebroventricular administration and to be truly epileptic in small rodents, although about 8 h after metaphit administration, the power spectra increased and was more intense in the period of sound onset and seizure events. Taken together, DSIP makes an optimal ratio between inhibitory and excitatory amino acid neurotransmitters and may represent one of the endogenous control systems of the brain, thus exerting the protective effect against the seizures. The results obtained throughout the present study corroborate and broaden the data on prolonged antiepileptic DSIP effect. CONCLUSION: The results of the present study strongly suggest that treatment of adult rats with DSIP and its analogue DSIP1-4 should be considered as potential natural antiepileptics.