Behavioral and Gene Regulatory Responses to Developmental Drug Exposures in Zebrafish

Developmental consequences of prenatal drug exposure have been reported in many human cohorts and animal studies. The long-lasting impact on the offspring—including motor and cognitive impairments, cranial and cardiac anomalies and increased prevalence of ADHD—is a socioeconomic burden worldwide. Identifying the molecular changes leading to developmental consequences could help ameliorate the deficits and limit the impact. In this study, we have used zebrafish, a well-established behavioral and genetic model with conserved drug response and reward pathways, to identify changes in behavior and cellular pathways in response to developmental exposure to amphetamine, nicotine or oxycodone. In the presence of the drug, exposed animals showed altered behavior, consistent with effects seen in mammalian systems, including impaired locomotion and altered habituation to acoustic startle. Differences in responses seen following acute and chronic exposure suggest adaptation to the presence of the drug. Transcriptomic analysis of exposed larvae revealed differential expression of numerous genes and alterations in many pathways, including those related to cell death, immunity and circadian rhythm regulation. Differential expression of circadian rhythm genes did not correlate with behavioral changes in the larvae, however, two of the circadian genes, arntl2 and per2, were also differentially expressed at later stages of development, suggesting a long-lasting impact of developmental exposures on circadian gene expression. The immediate-early genes, egr1, egr4, fosab, and junbb, which are associated with synaptic plasticity, were downregulated by all three drugs and in situ hybridization showed that the expression for all four genes was reduced across all neuroanatomical regions, including brain regions implicated in reward processing, addiction and other psychiatric conditions. We anticipate that these early changes in gene expression in response to drug exposure are likely to contribute to the consequences of prenatal exposure and their discovery might pave the way to therapeutic intervention to ameliorate the long-lasting deficits.

Alterations in Excitatory and Inhibitory Synaptic Development Within the Mesolimbic Dopamine Pathway in a Mouse Model of Prenatal Drug Exposure

The rise in rates of opioid abuse in recent years in the United States has led to a dramatic increase in the incidence of neonatal abstinence syndrome (NAS). Despite improved understanding of NAS and its acute symptoms, there remains a paucity of information regarding the long-term effects of prenatal exposure to drugs of abuse on neurological development. The primary goal of this study was to investigate the effects of prenatal drug exposure on synaptic connectivity within brain regions associated with the mesolimbic dopamine pathway, the primary reward pathway associated with drug abuse and addiction, in a mouse model. Our secondary goal was to examine the role of the Ca+2 channel subunit α2δ-1, known to be involved in key developmental synaptogenic pathways, in mediating these effects. Pregnant mouse dams were treated orally with either the opioid drug buprenorphine (commonly used in medication-assisted treatment for substance use patients), gabapentin (neuropathic pain drug that binds to α2δ-1 and has been increasingly co-abused with opioids), a combination of both drugs, or vehicle daily from gestational day 6 until postnatal day 11. Confocal fluorescence immunohistochemistry (IHC) imaging of the brains of the resulting wild-type (WT) pups at postnatal day 21 revealed a number of significant alterations in excitatory and inhibitory synaptic populations within the anterior cingulate cortex (ACC), nucleus accumbens (NAC), and medial prefrontal cortex (PFC), particularly in the buprenorphine or combinatorial buprenorphine/gabapentin groups. Furthermore, we observed several drug- and region-specific differences in synaptic connectivity between WT and α2δ-1 haploinsufficient mice, indicating that critical α2δ-1-associated synaptogenic pathways are disrupted with early life drug exposure.

Alterations in excitatory and inhibitory synaptic development within the mesolimbic dopamine pathway in a mouse model of prenatal drug exposure

The rise in rates of opioid abuse in recent years has led to an increase in the incidence of neonatal abstinence syndrome (NAS). Despite having a greater understanding of NAS and its symptoms, there still remains a lack of information surrounding the long-term effects of prenatal exposure to drugs of abuse on neurological development. One potential outcome of prenatal drug exposure that has been increasingly explored is disruption in normal synaptogenesis within the central nervous system. Both opioids and gabapentin, an antiepileptic drug commonly co-abused by opioid abuse disorder patients, have been shown to interfere with the normal functioning of astrocytes, non-neuronal glial cells known to serve many functions, including regulation of synaptic development. The goal of this study was to investigate the effects of prenatal drug exposure on synaptogenesis within brain regions associated with the mesolimbic dopamine pathway, the primary reward pathway within the brain associated with drug abuse and addiction, in a pregnant mouse model. Immunohistochemistry (IHC) and confocal fluorescence microscopy imaging studies on the brains of postnatal day 21 (P21) mouse pups revealed a significant increase in the mean number of excitatory synapses within the anterior cingulate cortex (ACC), nucleus accumbens (NAc), and prefrontal cortex (PFC) in mice that were prenatally exposed to either the opioid drug buprenorphine or gabapentin. These studies also revealed a significant decrease in the mean number of inhibitory synapses within the NAc and PFC of mice treated with buprenorphine. This observed net increase in excitatory signaling capability within the developing mesolimbic dopamine pathway suggests that exposure to drugs of abuse in utero can trigger maladaptive neuronal connectivity that persists beyond the earliest stages of life.