Brief postnatal exposure to phenobarbital impairs passive avoidance learning and sensorimotor gating in rats
Introduction
Phenobarbital (PB) is the most commonly utilized drug for the treatment of neonatal seizures [1], [2], [3] despite growing concerns about its efficacy [4], [5] and safety in neonatal or infant populations. For example, prolonged early-life exposure to phenobarbital as a treatment for febrile seizures has been associated with reduced IQ [6], [7]. Comparable studies examining shorter exposures have not been performed.
Preclinically, there is mounting evidence that even brief exposure to phenobarbital during early postnatal life can have long-lasting effects on brain development in rodents. For example, when given even once to postnatal day (P)7 rats, phenobarbital induced a profound increase in neuronal apoptosis throughout a variety of cortical (e.g., frontal and parietal cortices) and subcortical (e.g., hippocampus, nucleus accumbens, amygdala, and thalamus) structures [8], [9], [10]. This effect has been well documented by several groups, with the period of vulnerability lasting until ~ P10–P14 [8]. Early-life phenobarbital exposure is also associated with changes in the cortical proteome, including genes associated with synaptic function and regulation of oxidative stress [11].
Importantly, P7 exposure to PB also induces changes in nervous system function. For example, between P10 and P18, there is normally a robust increase in the number of functional excitatory and inhibitory synapses in the striatum [12]. By contrast, when rats were exposed to PB on P7, striatal synaptic development was stunted [12], [13]. Interestingly, when the timing of PB exposure was shifted to P10, normal maturation patterns were found [12].
We [12], [13], [14], [15], [16] and others [17], [18], [19], [20], [21] have also reported functional effects of early-life PB exposure on adult behavior. One of the most consistent findings is impaired spatial memory in PB-exposed animals tested as adults; exposure from P6 to P10, from P7 to P14, or from P2 to P21 all disrupted adult spatial memory in the Morris water maze or radial arm maze [15], [17], [20], [21]. Deficits in other memory tasks (i.e., delayed alternation [21], fear conditioning [13], [15], reversal learning [12]) have also been reported after PB exposure in early life. Additional behavioral changes following acute or subacute neonatal exposure include impaired prepulse inhibition (PPI) [13], [14], [15], hypersensitivity to the locomotor-enhancing effects of amphetamine [14], [16], decreased anxiety-like behavior, and reduced social exploration [15].
The present study had two objectives. The first objective was to better approximate a clinical schedule of PB exposure. Previous studies have used either acute [12], [14] or prolonged [13], [15], [18], [20], [21] exposure to PB. While a single exposure is useful for examining the “worst-case” scenario of drug toxicity (i.e., even a single dose is sufficient to alter behavior), it does not mirror clinical recommendations [3]. Conversely, longer exposures can exceed both the therapeutically relevant dose (due to drug accumulation) and the developmentally equivalent [22] time period during which treatment would occur. To reduce these confounds, here, we examined the effects of subacute administration of PB (P7, P8, and P9) on subsequent adult behavior. Pups received a loading dose on P7 and half doses on P8 and P9 to avoid drug accumulation.
The second objective of this study was to examine a previously unexplored behavioral domain: psychostimulant reinforcement. We chose this measure because of the enhanced locomotor response PB-exposed rats display to psychostimulants [14], [16] and because of the profound apoptosis that occurs in limbic structures that mediate reward [10]. As a basis for comparison, PPI was also examined, which is impaired by both acute and chronic exposures and, thus, serves as a positive control for the present study [13], [14], [15]. We also chose a step-through passive avoidance task as a measure of associative learning. Associative learning is impaired by chronic exposure [13], [15] but has yet to be examined after acute or subacute exposure.
Section snippets
Animals
Timed-pregnant Sprague–Dawley rats (Harlan Laboratories, Frederick, Maryland) were housed in the Georgetown University Division of Comparative Medicine. Animals were maintained in a temperature-controlled room (21 °C) with a 12-h light cycle (0600–1800 lights on). Food and water were available ad libitum. A total of 30 pups (a mix of male and females) were used, and date of parturition was designated P0 for all pups. Treatment was counterbalanced across litters and sex, and all manipulations
Body weight
Control and PB groups had equivalent mean body weight before the onset of treatment (14.6 g). Rats treated with PB showed a mean 2% increase in body weight from P7 to P8 and a 9% increase from P7 to P9. By contrast, vehicle-treated animals showed a 15% increase from P7 to P8 and a 34% increase from P7 to P9. Two-way analysis of variance revealed a significant main effect of age (postnatal day) [F2,64 = 102.7, P < 0.0001], a significant main effect of treatment [F1,32 = 15.34, P = 0.0004], and a
Discussion
Here, we found that subacute, clinically relevant neonatal exposure to phenobarbital resulted in behavioral alterations detected in adults.
Consistent with our previous studies, PB exposure was associated with deficits in prepulse inhibition. Prepulse inhibition is a measure of sensorimotor gating that is critically dependent on the limbic forebrain structures, including the hippocampus, amygdala, and nucleus accumbens [30]. The fact that this phenotype is highly consistent across studies may be
Disclosures
None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References (41)
- et al.
Neonatal seizures: multicenter variability in current treatment practices
Pediatr Neurol
(2007) - et al.
Neonatal seizures: treatment practices among term and preterm infants
Pediatr Neurol
(2012) - et al.
Uncoupling of EEG-clinical neonatal seizures after antiepileptic drug use
Pediatr Neurol
(2003) - et al.
Brief alteration of NMDA or GABAA receptor-mediated neurotransmission has long term effects on the developing cerebral cortex
Mol Cell Proteomics
(2008) - et al.
Neonatal exposure to phenobarbital potentiates schizophrenia-like behavioral outcomes in the rat
Neuropharmacology
(2012) - et al.
Long-term reduction in spontaneous alternations after early exposure to phenobarbital
Int J Dev Neurosci
(1984) - et al.
Long term reduction in eight arm maze performance after early exposure to phenobarbital
Int J Dev Neurosci
(1985) - et al.
Hippocampal cholinergic alterations and related behavioral deficits after early exposure to phenobarbital
Brain Res Bull
(1992) - et al.
Comparative aspects of the brain growth spurt
Early Hum Dev
(1979) - et al.
Anticonvulsant effects of phenobarbital and primidone during ontogenesis in rats
Epilepsy Res
(1991)
The central amygdala is involved in conditioning but not in retention of active and passive shock avoidance in male rats
Behav Neural Biol
Modulation of anxiety-related behaviours following lesions of the prelimbic or infralimbic cortex in the rat
Brain Res
Guidelines on neonatal seizures
Neonatal seizures: after all these years we still love what doesn't work
Neurology
Phenobarbital for febrile seizures—effects on intelligence and on seizure recurrence
N Engl J Med
Late cognitive effects of early treatment with phenobarbital
Clin Pediatr (Phila)
Antiepileptic drugs and apoptotic neurodegeneration in the developing brain
Proc Natl Acad Sci U S A
Antiepileptic drugs and apoptosis in the developing brain
Ann N Y Acad Sci
Pattern of antiepileptic drug-induced cell death in limbic regions of the neonatal rat brain
Epilepsia
Neonatal exposure to antiepileptic drugs disrupts striatal synaptic development
Ann Neurol
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- 1
These authors contributed equally.
- 2
Current address for Dr. Kondratyev is National Institutes of Health, Center for Scientific Review. This work was prepared while AK was employed at GU. The opinions expressed in this article are the author's own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government.