Jennifer L. van de Kamp

Prenatal nicotine alters nicotinic receptor development in the mouse brain

Maternal smoking during pregnancy may affect development of the child, but little is known about potential mechanisms of these effects. Since chronic nicotine treatment alters brain nicotinic receptors in adults and also evokes tolerance which is regulated by genetic factors, pregnant mice of two inbred strains underwent chronic nicotine infusion to determine whether the developmental pattern of mouse brain nicotinic receptors would be altered. C3H/2ibg and C57BL/6ibg mice were infused SC with saline or 2.0 mg/kg/h nicotine during the last half of pregnancy. The developmental profiles of [3H]nicotine and alpha-[125I]bungarotoxin binding in seven brain regions obtained from the offspring were measured. Prenatal nicotine treatment increased levels of [3H]nicotine binding at birth in the C3H hypothalamus, hippocampus, and possibly the cortex, and in the C57BL cortex. At later ages (20-30 days), [3H]nicotine binding was elevated in the C3H hindbrain, hippocampus, striatum, midbrain, and possibly the cortex. The C57BL hindbrain, hippocampus, midbrain, and cortex also showed increased binding at 20-30 days. Little, if any, effect of prenatal nicotine treatment was observed on the development of the alpha-[125I]bungarotoxin binding site. Since upregulated [3H]nicotine binding returns to control levels in adult animals within seven days following termination of chronic nicotine infusion, it is unlikely that simple upregulation is responsible for the changes observed in 20-30-day-old mouse brains.

Substance P facilitation of memory: Effects in an appetitively motivated learning task

Food deprived, heterogeneous strain (HS/IBG) mice were trained on two different discrimination tasks for food reinforcement. In one experiment animals were trained to make spatial discriminations in a T maze. Immediately after training they were given subcutaneous injections of either substance P (1 ng/g) or vehicle. Twenty-four hours later the animals were given reversal training in the same maze. The results showed that substance P-treated animals took significantly longer to acquire the reversal habit than did control mice. In a second experiment, animals were trained to make visual discriminations in a T maze. Immediately after reaching acquisition criterion animals were injected with either substance P (1 ng/g) or vehicle. Different groups of mice were retrained on the same task either 1, 2, 3, or 7 days after original learning. Savings scores were calculated and, at every interval, substance P-treated mice retained the task better than control animals. One interpretation of these data is that substance P-treated mice remembered the original task significantly better than vehicle-injected control animals.

Sensitivity of inbred and selectively bred mice to ethanol.

The Long-Sleep (LS) and Short-Sleep (SS) mice were bred for differences in sensitivity to ethanol as measured by duration of loss of the righting response (sleep time). The foundation population was a heterogeneous stock (HS) which was derived from a cross of eight inbred strains. Ethanol-induced sleep time and waking blood and brain ethanol levels were measured in the eight inbred strains, LS, SS and HS mice. The C3H and ISBI strains were quite resistant to ethanol as measured by sleep time, and only one, RIII, was very sensitive. Waking ethanol concentrations were similar for all of the inbreds, implying a narrow range of central nervous system sensitivity to ethanol. The HS mice had relatively short sleep times and blood ethanol levels equal to most of the inbred. The LS mice were significantly more, and the SS mice significantly less sensitive to ethanol than any of the inbreds or HS mice. These studies suggest that the extremes of CNS sensitivities to ethanol manifested by the LS and SS mice cannot be traced to any of the inbred strains, and must have arisen through the selection process by changes in allelic frequencies of those genes conferring ethanol sensitivity and resistance.

Developmental profile of hepatic alcohol and aldehyde dehydrogenase activities in long-sleep and short-sleep mice

Ethanol is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (ADH). The product, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (AHD). Mouse liver contains five major constitutive AHD isoenzymes: mitochondrial high Km (AHD-1), mitochondrial low Km (AHD-5), cytosolic high Km (AHD-7), cytosolic low Km (AHD-2) and microsomal high Km (AHD-3). The Long-Sleep (LS) and Short-Sleep (SS) mice differ in their sleep time response to ethanol as early as 10 days of age, and this difference increases with increasing age. Age- and genotype-related differences in metabolism could account for the pattern of responses seen in these mice. We measured the activity of hepatic ADH and the five AHD isoenzymes in LS and SS mice from 3 days of age to adulthood to determine if there were differences in the developmental profiles of these enzyme activities. We found no sex differences in the developmental profile of either ADH or AHD, and the LS and SS mice have nearly identical ADH and AHD activities with the possible exception of the high Km mitochondrial enzyme activity between days 3 and 6, and the low Km mitochondrial enzyme between days 28 and 32. Thus, it appears that differences in ethanol or acetaldehyde metabolism do not contribute significantly to the differential sensitivity to ethanol between young LS and SS mice or to the differential sensitivity between young and adult mice.

Species differences in diisopropylfluorophosphate-induced decreases in the number of brain nicotinic receptors

DBA and C3H mice were injected chronically with 2.0 mg/kg diisopropylfluorophosphate (DFP) every other day for 2 or 4 weeks. Although acetylcholinesterase (AChE) activity and muscarinic receptor numbers ([3H] quinuclidinyl benzilate (QNB) binding) were decreased in DFP-treated DBA and C3H mice, the number of nicotinic receptors (L-[3H]nicotine and alpha-[125I]bungarotoxin (BTX) binding) was unchanged by chronic DFP treatment. Sprague-Dawley rats injected chronically with lower doses of DFP than were used in mice exhibited a greater reduction in AChE activity, as well as accompanying decreases in [3H]QNB and [3H]nicotine binding. Neither species exhibited changes in alpha-[125I]BTX following chronic DFP injection. The effects of chronic DFP treatment on sensitivity to DFP and to nicotine were also assessed in the two mouse strains using a battery of behavioral and physiological tests that included rotarod performance, Y-maze crossing and rearing activity, heart rate, and body temperature. No tolerance to DFP was observed in either mouse strain after 2 weeks of treatment. Following 4 weeks of treatment, DFP-treated DBA mice exhibited modest tolerance to the effect of DFP on body temperature. C3H mice did not survive the 4-week treatment. Some evidence for reduced sensitivity to nicotines effects was detected in the DFP-treated DBA mice, but cross-tolerance to nicotine was not observed in the DFP-injected C3H mice. Because chronic DFP treatment did not evoke a change in the number of brain nicotinic receptors, the reduced sensitivity to some of nicotines effects seen in DBA mice must be due to some factor other than receptor downregulation.

Alterations in brain catecholamines during pregnancy

During pregnancy mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The potential role of brain catecholamines in mediating this behavior was examined in the present study. The concentration and turnover of norepinephrine (NE) and dopamine (DA) were measured in hippocampus, striatum, midbrain and cortex in control, pregnant and delivery-day mice. There were no significant changes from control in DA levels during pregnancy and parturition. The turnover of DA was not altered during pregnancy, except for a small increase in turnover rate in the hippocampus. The concentration of NE decreased during pregnancy, and rose at parturition. This effect was most striking in the hippocampus. The turnover of NE was markedly depressed during pregnancy, with the hippocampus again being most affected. These data imply a role for NE, but not DA in the mediation of increased seizure susceptibility during pregnancy.

The effect of naloxone administration on pregnancy-associated seizures

Pregnant mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The possibility that the well-known increase in beta-endorphin concentration which accompanies pregnancy was involved in this effect was examined by testing whether naloxone administration could block the increased seizure susceptibility. Pregnant female, control female and male C3H mice were treated with 5-50 mg/kg naloxone 5 min before flurothyl seizure testing. Naloxone markedly increased clonic seizure susceptibility in all three groups at a dose of 50 mg/kg, but had little effect at lower doses. In contrast, naloxone had differential effects on myoclonic seizures in pregnant and control female mice, being anticonvulsant in the controls, but proconvulsant in the pregnant mice. A role for endogenous opiates is unlikely in mediating clonic seizures in pregnant mice, but may be involved in myoclonic seizures.

Relationships between Pregnancy and Vitamin B-6 Nutriture on Brain 3-Hydroxykynurenine Concentrations in Mice.

The kynurenine (KYN) pathway of tryptophan metabolism produces several neuroactive metabolites, including 3-hydroxykynurenine (3HK). The pathway is subject to regulation by a number of effectors including pregnancy and availability of vitamin B-6. Vitamin B-6 depleted humans and animals excrete abnormally high concentrations of KYN metabolites in urine. In pregnancy, vitamin B-6 deficiency is commonly seen, and tryptophan metabolism is often found to be altered. We measured concentrations of 3HK in brains of DBA/2Ibg and A/Ibg mice as functions of pregnancy and dietary level of vitamin B-6. Pregnant DBA mice are more susceptible to flurothyl-induced seizures than controls, pregnant A mice are not. Significant elevations of 3HK were found in brains of pregnant mice, and the increases were greater in the pregnancy-associated seizure prone DBA, than in the A mice. In the A mice, brain 3HK concentrations were negatively correlated with dietary vitamin B-6 levels, as expected; however, in the DBA mice these correlations were positive, indicating an unusual response to vitamin B-6 restriction. The accumulation of a cytotoxic, excitatory metabolite, 3HK, in brain may contribute to the increased seizure susceptibility of susceptible pregnant mice, perhaps though its effects as an endogenous modulator of excitatory amino acid receptor systems.