Rachelle H.B. Fishman

Long-lasting effects of early barbiturates on central nervous system and behavior.

Forty years of prescribing barbiturates to pregnant women and infants, and thirty years of animal research have shown that barbiturates affect the developing central nervous system (CNS) and behavior. This paper compiles and reviews animal and selected human literature in this research area. Early barbiturate exposure in animals reduces brain weight with related changes in brain biochemistry and neuromorphology. Significant changes may be found in surviving adult offspring. Evidence of CNS and behavioral damage in human beings due to early barbiturate exposure is not clearcut, however, confounded by the conditions for which the drugs are prescribed. In animals, early drug exposure significantly reduces levels of hormones, vitamins, and other biologically active macromolecules via (long-lasting) induction of hepatic metabolizing enzymes. Whether or not in humans treated with barbiturates, hormone levels remain within the normal range (by-feed-back regulation) and, also, if vitamin deficiencies can be simply corrected by supplements is still being debated. Early barbiturates administered to animals is associated with long-lasting disturbances in activity, learning performance, sexual behavior, and reproductive function, but not in a simple dose-exposure related manner. Animal studies show that long-lasting functional tolerance to drugs develops following early barbiturate exposure. Although infants become passively addicted following in utero exposure, there is as yet no data on subsequent development of human adult tolerance. Drug related damage must, in any case, be weighed against therapeutic benefits of drug administration and the results of failure to treat.

Early barbiturate treatment eliminates peak serum thyroxine levels in neonatal mice and produces ultrastructural damage in the brains of adults.

Serum thyroxine levels peak sharply at the end of the second postnatal week in mice. Treating neonatal mice with barbiturates (PhB) completely eliminates this marked thyroxine (T4) peak without significantly affecting nonpeak levels at other ages. PhB treatment at this same time also establishes or induces long-lasting degenerative processes that continue to result in ultrastructural neural deficits in adults, long after treatment has been discontinued. Similar neural deficits have been observed in animals which were deficient in T4 during this same period of development.

Ultrastructural evidence of long-lasting cerebellar degeneration after early exposure to phenobarbital in mice.

Previous studies in this laboratory demonstrated a 20 to 30% reduction in cerebellar Purkinje and granule cells after exposure to phenobarbital (PhB) early in life. Therefore, neurons in the cerebellar cortex were examined for signs of cytologic degeneration using transmission electron microscopy (TEM) after exposure to PhB pre- and postnatally. Pregnant mice were given the acid form of PhB in their milled food (3 g/kg, gestation days 9 to 18) and water, ad libitum. Neonates were injected s.c. with an aqueous solution of sodium PhB (50 mg/kg body weight), days 2 to 21 after delivery. Controls were fed regular food or injected with the vehicle. The offspring were anesthetized on day 14 or 50 by an acute overdose of PhB and immediately perfused with a formaldehyde-paraformaldehyde or glutaraldehyde solution. The pyramis vermis of the cerebellar cortex was excised and processed routinely for TEM. The three layers of the cortex were examined. A short-term effect (at day 14) was found. More significantly, the treatment appeared to establish or trigger a degenerative process, the results of which were still apparent at day 50, more than 30 days after the termination of PhB treatment. Using double-blind evaluation for the presence and frequency of abnormalities, the cerebellar neurons of treated animals had 155 to 300% more abnormalities compared with control animals. Abnormalities included (i) Mitochondrial degeneration, ranging from swelling, collapse of cristae, vacuolization, to total granularization; (ii) lamellar bodies distributed throughout the cytoplasm and in cell processes; and (iii) myelin sheath degeneration, including periodic swelling and collapse, twisting of the coat, and scattered, unevenly stained areas. Damage was usually focal. Affected cells were found adjacent to normal cells in all areas of the cortex. PhB may cause the neural damage through a possible hormonal role.

CORRELATED ULTRASTRUCTURAL DAMAGE BETWEEN CEREBELLUM CELLS AFTER EARLY ANTICONVULSANT TREATMENT IN MICE

The anticonvulsants phenobarbital and diphenylhydantoin administered early in life to mice resulted in significant and long‐lasting ultrastructural damage, including abnormalities of mitochondria, myelin sheaths and lamellar inclusion bodies inside identified cells throughout the cortical layers of the cerebellum in treated vs control mice. The magnitude, distribution and duration of damage was age and treatment specific. No differences were detected in density of parallel fiber processes nor in synapse density within the molecular layer. Neuron profiles containing damaged organelles were not homogeneously distributed but made up only a small fraction of the total cell population examined. In our experiments, there was an overall within‐animal correlation explaining 45% of the magnitude of damage in different cerebellar regions, but between synaptically connected cells, specifically mossy fiber axon varicosities and granule cell dendrite profiles, the subset population ratio of damaged‐to‐total mitochondria was highly significantly correlated (70–90%; P<0.001). We hypothesized that some correlated transneuronal degeneration and death in the central nervous system may have a transynaptically regulated component that first appears as correlated damage between synaptically connected cells, perhaps regardless of the degree of toxicity. The orderly cytoarchitecture and cell connections of the cerebellar cortex can be used to study these patterns of degeneration.

Resistance to barbiturate is changed by developmental alteration of dopamine receptor sensitivity

Adult rats whose dopamine receptor number was reduced via prenatal haloperidol exposure were less resistant than controls to pentobarbital narcosis. This altered resistance was functional (CNS) and not metabolic, as shown by pentobarbital brain levels. These results are consistent with our previous experiments, indicating that changes in the number of dopamine receptors may underlie relative susceptibility to barbiturates.

Regional brain superoxide dismutase activity is altered differently by heat in warm and cool mice

The significant regional variation in brain superoxide dismutase (SOD) activity was similar in mice from both warm and cool cohorts. Mice in the cool cohort generally had higher SOD activity, which varied significantly with body temperature in striatum and in preoptic area of the hypothalamus. Changes in SOD activity following heating were revealed only when warm and cool cohorts were analysed separately. SOD activity decreased significantly in striatum, hypothalamus, and hippocampus of the cool cohort only. The decline was to levels consistent with those of the warm cohort. Body temperature of cool mice increased more than that of warm mice following each increment of heating so resultant body temperatures became similar. The role of SOD as part of a differential defense against heat stress in warm and cool mice is presented.

The modulation of transmitter release in motor nerve endings varies witthe type of muscle fiber innervated

Electrophysiological studies of synaptic transmission in mammalian muscles revealed substantial differences between neuromuscular synapses associated with fast versus slow-twitch motor units. Synapses of fast units exhibited much higher tetanic and post-tetanic potentiation of neurotransmitter release than those of slow units. On the other hand, the tetanic potentiation of slow units increased faster as a function of the frequency of activation than that of fast units. These differences suggest a possible specialization of neuromuscular synapses with the type of motor units they are associated with.

Developmental manipulations of dopamine receptors modulate sensitivity to barbiturate

Developmental Manipulations of Dopamine Receptors Modulate Sensitivity to Barbiturate Yanai, J. and Fishman, R.~.B. The Hebrew University-Hadassah Medical School, Department of Anatomy & Embryology, 91010 Jerusalem, Israel. In our previous experiments, when post-synaptic dopamine (DA) receptors of adult mice were made supersensitive, they possessed a lesser CNS sensitivity to ethanol and barbiturate. A reciprocal and complementary experiment was conducted. Pregnant sabra rats received twice daily S.C. injection of 2 mg/kg halperidol (H) on gestation days 14 to 18, in order to induce DA receptors subsensitivity in the treated offspring. Control and H offspring were tested at adulthood for pentobarbital, (PenB; 40mg/kg) induced sleep time. H females and males slept 43% and 81% longer than their controls, respectively (p<O.001), indicating greater sensitivity to PenB in H offspring. That this increased sensitivity was functional (CNS) is suggested by the lower brain PenB levels than controls upon awakening among H animals; 26% decrease in females and 18% in males (p<O.05), respectively. The results confirm the hypothesis that brain sensitivity to barbiturate is negatively correlated with the sensitivity of DA receptors.

Thermal responses and survival after heat exposure are modulated by maintained differences in body temperature in mice

When individual mice were examined, it was found that the colonic body temperatureTcol of each individual within a genetically heterogeneous population tended to remain either above (“warm”) or below (“cool”) the population mean.Tcol of warm, but not cool, mice showed circadian variation. When exposed to aTa of 43° C, theTcol of cool mice increased by as musch as 2.4° C more than that of warm mice for a given 15 min increment of heating at 43°C. Survival of mice after acute lethal heat load (LD75, −45°C) was significantly inversely correlated withTcol. Small persistent differences in body temperature of individuals may indicate differing thermal adaptedness.

Diethystilbesterol counteracts barbiturate narcosis and hypothermia in male mice

Treatment with DES one hour prior to pentobarbital injection resulted in diminution of the narcotic sleep and hypothermia usually found after pentobarbital (50 mg/kg 1) injection in male mice. The effect was biphasic: significantly countered relative to saline pretreated controls at low (.001-.10 mg/kg 1) and at high (10-50 mg/kg -1) DES doses only. Giving DES alone did not change core body temperature compared to saline injected controls, at either 25 degrees C or at 33 degrees C. At 33 degrees C, neither PeB narcosis nor body temperature loss was significantly inhibited by DES. Possible cytoplasmic, nonsex differentiated bases for these estrogen effects on barbiturate action in the brain is discussed.