Richard W. Keller

Prenatal cocaine exposure.

Abstract: Cocaine abuse is a significant problem not only in the general population but also among pregnant women. Since cocaine readily crosses the placenta and is metabolized slowly in fetuses, they can be exposed to significant levels of cocaine for long periods. In humans the most common consequences of cocaine abuse during pregnancy include premature birth, lower birth weight, respiratory distress, bowel infarctions, cerebral infarctions, reduced head circumference, and increased risk of seizures. Behaviorally these new‐borns show an increased degree of “tremulousness,” crying and irritability, and are over‐reactive to environmental stimuli. Within a month these behaviors have recovered dramatically, but not to normal levels. Thus while there are a number of abnormalities associated with cocaine‐exposed neonates, they are not imminently debilitating or life‐threatening. However, the long‐term consequences of this prenatal cocaine exposure remain to be elucidated. We have examined a rat model for neurochemical, neuroanatomical and behavioral changes resulting from prenatal cocaine exposure. Since cocaine is known to act by blocking the inactivation of the neurotransmitters dopamine, serotonin and norepinephrine, our studies have focused on brain dopamine (DA) and serotonin (5‐HT) pathways. In this model system we have found neurochemical changes that are present at birth and that return to normal as the rat ages‐similar to the recovery observed in infants. However, there are other neurochemical, anatomical and behavioral changes that persist after birth which may provide insights into the long‐term consequences. It is hoped that by understanding the changes occurring in this rat model we will be better prepared to devise pharmacological interventions to circumvent the secondary consequences of prenatal cocaine exposure. These consequences might include increased susceptibility to drug addiction, seizures, depression, schizophrenia, Parkinsons disease, etc.

Side and region dependent changes in dopamine activation with various durations of restraint stress

Exposure to various mild stressors has been shown to result in the activation of dopamine containing neuronal systems projecting to the medial prefrontal cortex (PFC), to a lesser extent the nucleus accumbens septi/olfactory tubercle (NAS) and, in a few studies, the striatum. It has also been shown that dopamine (DA) systems on different sides of the PFC are successively activated as stressors are prolonged. We have therefore examined the effects of variation in the duration of a restraint stressor (15, 30 and 60 min) on region and side dependent alterations in DA utilization in the PFC, NAS and striatum. Increases in the concentrations of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and/or homovanillic acid (HVA) or in their ratios with DA were seen in all regions examined with the largest effects occurring in the PFC and lesser effects in the NAS and striatum. In each region, the magnitude of these effects varied with time of restraint exposure. In the PFC, lateralized alterations in HVA and DA were seen over time with effects progressing from a left greater than right involvement at 15 min to a right greater than left involvement at 60 min. These results are discussed with reference to side and region dependent effects on brain DA systems as stressors are prolonged and the implications they may have for lateralized regional brain activity associated with stressor precipitated psychiatric disease.

Left and right 6-hydroxydopamine lesions of the medial prefrontal cortex differentially alter subcortical dopamine utilization and the behavioral response to stress

The effects of left and right prefrontal cortical dopamine (DA) depletion on circling behavior, stress-escape behavior and subcortical DA function were examined in rats exhibiting left or right turning biases. 6-Hydroxydopamine lesions of the medial prefrontal cortex (PFC) caused significant DA depletions when assessed in separate studies at 3 days and 3-4 weeks. However, depletions were smaller at 3-4 weeks and there was a significant increase in DA concentration on the left side following right lesions. Significant increases in striatal DA content were observed following lesions of either side at 3-4 weeks, but not at 3 days. No changes in DA concentration were observed in the nucleus accumbens septi (NAS). Left circling rats significantly increased their circling behavior following right sided lesions and showed disrupted footshock-escape behavior following left sided lesions. Performance of the footshock-escape task exerted an effect on striatal and NAS DA utilization as indicated by the ratio of 3,4-dihydroxyphenylacetic acid (DOPAC) to DA. The effects of footshock on NAS DA utilization were greater following left PFC lesions as compared to the right lesion and sham conditions. These lesion effects were also greater in left- than in right-turning animals. The data indicate that an intrinsic asymmetry in brain DA systems interacts with left and right PFC lesions to differentially determine subcortical DA function and behaviors that it subserves.

Prenatal cocaine increases striatal serotonin innervation without altering the patch/matrix organization of intrinsic cell types

The effect of prenatal cocaine on the anatomical development of the striatum was examined. The distribution and density of dopaminergic innervation of the striatum of animals exposed to cocaine during the second and third week of gestation was not noticeably different from prenatally saline-injected or untreated controls at any age. The patch/matrix organization of the striatum also appeared unaltered: neurons exhibiting dense substance P staining were localized to patches that overlapped dopamine terminal patches early in development, and enkephalin- and calbindin-immunoreactive neurons were found segregated to the matrix. Histochemical staining for acetylcholinesterase and NADPH diaphorase also revealed no differences between prenatally cocaine-treated and control brains. Whereas prenatal cocaine treatment failed to modify the basic compartmental organization of the striatum, it did lead to a hyperinnervation of serotonin-immunoreactive fibers which developed slowly after birth. Thus prenatal exposure to cocaine is capable of altering the ingrowth of serotonergic projections to the striatum while producing no change in the organization of neurons intrinsic to the striatum.

Enhanced cocaine self-administration in adult rats prenatally exposed to cocaine

Rats that had been prenatally exposed to either cocaine or saline were examined as adults using continuous reinforcement (FR1) cocaine self-administration. Initially these rats were water-deprived and trained to bar-press for water; no differences across prenatal treatments were observed during this training phase. After complete rehydration and implantation of an intravenous cannula into the external jugular vein, animals were introduced to cocaine self-administration with a nocturnal and subsequent 3 h exposure. During daily test sessions rats were allowed to self-administer cocaine for 1 h/day. Prenatal cocaine exposure led to a marked and stable enhancement of the rates of self-administration for up to 13 days, the longest time point examined. These results suggest that prenatal cocaine exposure can alter cocaine reinforcement in adult animals.

Chronic antidepressant drug treatment reduces turning behavior and increases dopamine levels in the medial prefrontal cortex.

The effects of chronic administration of the antidepressant drugs desipramine, nortryptiline and paroxetine (PAR) (10 mg/kg/day, 21 days) on changes in turning (circling) behavior and on norepinephrine (NE), dopamine (DA) and serotonin and their metabolites 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid (5-HIAA) in the medial prefrontal cortex (PFC), nucleus accumbens and striatum were evaluated in rats. All three drugs eliminated turning biases in right turning rats. All drugs increased DA concentrations in the PFC while PAR increased NE in the PFC and reduced 5-HIAA in all three structures. The results are discussed with reference to previous findings involving brain asymmetry in depression.

Prenatal cocaine alters later sensitivity to cocaine-induced seizures☆

Rats that had been prenatally exposed to cocaine were tested later in life for their sensitivity to cocaine-kindled seizures and acute cocaine-induced seizures. When treated daily with cocaine, beginning at one month of age, males prenatally exposed to 40 mg/kg cocaine developed seizures in a fewer number of days than those prenatally exposed to saline. Prenatally cocaine-treated females did not seize more rapidly than controls in the cocaine kindling paradigm; however, they were more susceptible to seizures in response to an acute high dose of cocaine. These results suggest that rats prenatally cocaine-treated are more sensitive to the seizure-producing effects of cocaine later in life, and this enhanced sensitivity is differentially expressed in males and females.

The neuroprotective properties of the superoxide dismutase mimetic tempol correlate with its ability to reduce pathological glutamate release in a rodent model of stroke

The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies have failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit the release of the excitotoxic amino acids glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine, and alanine. Microdialysate delivery of 10mM tempol reduced the amino acid release by 60-80%, whereas matching levels of edaravone had no effect. In line with these data, an intracerebroventricular injection of tempol but not edaravone (500 nmol each, 15 min before MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior at removing superoxide anion, whereas edaravone was more potent at scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggest that the neuroprotective properties of tempol are probably related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.

Regional expression of constitutive and inducible transcription factors following transient focal ischemia in the neonatal rat: influence of hypothermia.

Ischemia is a potent modulator of gene expression. Differential expression of transcription factors after focal ischemia may reflect the potential for neuronal recovery in peri-ischemic regions. Previously, we demonstrated that hypothermia reduces the volume of damage in a model of neonatal focal ischemia. In the present study, immunocytochemistry was used to assess the temporal and spatial profiles of the transcription factors Fos and pCREB under normal and hypothermic conditions in this neonatal model of focal ischemia. At 7 days of age, rat pups underwent a permanent middle cerebral artery occlusion (MCAo) coupled with a temporary 1-h occlusion of the common carotid artery (CCAo). They were maintained at 37 degrees C throughout ischemia and reperfusion (Normothermic), or given 1 h of hypothermic conditions (28 degrees C) either during the occlusion (Intraischemic Hypothermia) or during the second hour of reperfusion (postischemic hypothermia). In normothermic pups, Fos immunoreactivity peaked at early time points (4-8 h post-ischemia) in a narrow band in peri-ischemic regions. By later stages of reperfusion (12-24 h), there was a more widespread induction in peri-ischemic regions including the ipsilateral cortex. In contrast with Fos, the constitutive transcription factor pCREB was reduced in core regions at all time points examined. Both the c-fos induction in peri-ischemic regions and the reduction of pCREB in the core were attenuated by intraischemic hypothermia. Postischemic hypothermia altered the distribution of Fos immunoreactivity without significantly changing the number of Fos- and pCREB-immunoreactive cells compared to normothermic rats. Both intra- and postischemic hypothermia reduced the number of caspase-immunoreactive cells. Thus, focal ischemia in the P7 rat produces different distributions of Fos and pCREB than what has been observed in adult rats subjected to focal ischemia, and expression of these transcription factors can be altered by hypothermia.

Protective effects of brief intra- and delayed postischemic hypothermia in a transient focal ischemia model in the neonatal rat

Hypothermia provides neuroprotection in virtually all animal models of ischemia, including adult stroke models and the neonatal hypoxic-ischemic (HI) model. In these studies, brief periods of hypothermia are examined in a neonatal model employing transient focal ischemia in a 7-day-old rat pup. Pups underwent permanent middle cerebral artery (MCA) occlusion coupled with a temporary (1 h) occlusion of the ipsilateral common carotid artery (CCA). This study included five treatment groups: (1) normothermic (Normo)-brain temperature was maintained at 37 degrees C; (2) intraischemic hypothermia (IntraH)-28 degrees C during the 1-h ischemic period only; (3) postischemic hypothermia (PostH)-28 degrees C for the second hour of reperfusion only; (4) late-onset postischemic hypothermia (LPostH) cooled to 28 degrees C for the fifth and sixth hours of reperfusion only; and (5) Shams. After various times (3 days-6 weeks), the lesion was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) or hematoxylin and eosin (H&E) stains. Intraischemic hypothermia resulted in significant protection in terms of survival, lesion size, and histology. Postischemic hypothermia was not effective in reducing lesion size early after ischemia, but significantly reduced the eventual long-term damage (2-6 weeks). Late-onset postischemic hypothermia did not reduce infarct volume. Therefore, both intraischemic and postischemic hypothermia provided neuroprotection in the neonatal rat, but with different effects on the degenerative time course. While there were no observable differences in simple behaviors or growth, all hypothermic conditions significantly reduced mortality rates. While the protection resulting from intraischemic hypothermia is similar to what is observed in other models, the degree of long-term ischemic protection observed after 1 h of postischemic hypothermia was remarkable and distinct from what has been observed in other adult or neonatal models.