Research ArticleThird Ventricular Injection of CCL2 in Rat Embryo Stimulates CCL2/CCR2 Neuroimmune System in Neuroepithelial Radial Glia Progenitor Cells: Relation to Sexually Dimorphic, Stimulatory Effects on Peptide Neurons in Lateral Hypothalamus
Introduction
Recent studies have demonstrated that the neuroimmune system in the brain, functioning beyond the signaling for immune responses, produces profound changes in the development of neurons that result in long-term behavioral consequences (Cui et al., 2014, Crews et al., 2015, Chang et al., 2015, Chang et al., 2018). The wide-ranging and long-lasting functionalities of neuroimmune signaling are evident in the progression of ethanol’s effects on neuronal development in the embryo and subsequent changes in the offspring’s behavior associated with increased alcohol consumption. While chronic high doses or binge drinking episodes of ethanol are found to affect inflammatory pathways involving such molecules as Toll-like receptors which cause considerable neurodegeneration that exacerbates existing alcohol use disorder (Lippai et al., 2013, Crews and Vetreno, 2014, Flores-Bastias and Karahanian, 2018), there is evidence that lower levels and shorter periods of ethanol exposure can have very different effects. Along with elevated neuroimmune activity, these include an increase in cell proliferation, neurogenesis and migration (Camarillo and Miranda, 2008, Mooney and Miller, 2010, Skorput and Yeh, 2015) and in the expression and density of neurons in the lateral hypothalamus (LH) that express melanin-concentrating hormone (MCH) (Chang et al., 2015, Chang et al., 2018), a neuropeptide that promotes alcohol drinking and other behaviors associated with alcohol use disorder (Duncan et al., 2005, Morganstern et al., 2010, Karlsson et al., 2016). These studies of MCH in the LH demonstrate in the rat that maternal intraoral administration of ethanol, at low-to-moderate doses from embryonic day 10 (E10) to E15 during the period of peak hypothalamic neurogenesis (Ifft, 1972), increases in adolescent offspring the density of neurons that express the inflammatory chemokine C–C motif ligand 2 (CCL2) and its main receptor CCR2, a neuroimmune system also positively linked to alcohol intake (Blednov et al., 2005, June et al., 2015, Valenta and Gonzales, 2016). It also stimulates the co-expression of CCL2 and CCR2 in up to 90% of the MCH neurons (Chang et al., 2015, Chang et al., 2018)
The possibility that the CCL2/CCR2 system actually mediates these stimulatory effects of ethanol on MCH neurons is supported by the additional findings that they are similarly produced by maternal peripheral administration of CCL2 and are blocked by maternal administration of the CCR2 antagonist INCB3344 during the period of ethanol exposure (Chang et al., 2018, Chang et al., 2020a). A recent study of the developmental origins of this CCL2/CCR2-mediated stimulatory effect on neuronal development has shown that ethanol’s effects on MCH neurons that colocalize CCL2 and CCR2 in LH are evident in the embryo and neonatal offspring and are reversed by maternal administration of a CCL2 antibody that neutralizes endogenous CCL2 and a CCR2 antagonist that blocks CCL2’s main receptor (Chang et al., 2020a). Also in the embryo, maternal ethanol administration stimulates CCL2 cells dense in the hypothalamic neuroepithelium (NEP), a primary source of neurons along the third ventricle (Bedont et al., 2015), and it also increases the colocalization of CCL2 within radial glia progenitor cells and their laterally projecting processes, effects that are mimicked by maternal CCL2 administration and accompanied by an increased number of MCH neurons close to the radial glia cells and positioned along their processes (Chang et al., 2020b). Further tests demonstrate that these effects of maternal ethanol and CCL2 administration on the development of CCL2 and MCH neurons are consistently stronger in females than males, as shown in the embryo as well as adolescent offspring (Chang et al., 2018, Chang et al., 2020a, Chang et al., 2020b) Thus, in response to inflammatory challenges such as ethanol, these studies suggest the involvement of endogenous CCL2-rich radial glia progenitor cells in embryos in promoting, in a sexually dimorphic manner, the genesis and migration of MCH neurons toward their final destination in the LH.
While bringing attention to the CCL2/CCR2 system and radial glia in the NEP as a possible mechanism underlying these strong stimulatory effects on neuronal development, these studies with maternal peripheral manipulations of ethanol or CCL2 in pregnant rats leave open the question as to whether these alterations in this neuroimmune mechanism result from direct effects on the embryonic brain or whether they are indirect consequences of effects in the mother produced by peripheral manipulations. To investigate this question, we employed here in utero intracerebroventricular (ICV) injections to directly manipulate the embryonic brain during the period of peak hypothalamic neurogenesis, while having minimal impact on maternal neuroimmune factors. This ICV technique, allowing delivery of substances directly into the microenvironment of developing neural systems, not only avoids the confounding variables of maternal peripheral injections but also reduces some uncertainty regarding the molecular dynamics of neuroimmune compounds crossing the fetal blood brain barrier. While studies over the years have employed ICV injections to alter brain microenvironments in adult, adolescent, and postnatal rodents (Lewinski et al., 1984, Jung et al., 1994, Fang et al., 2013, Li et al., 2016), this technique has more recently been used in the embryo to administer genetic material with electroporation to induce localized changes in gene expression in both superficial and deeper embryonic brain regions (Walantus et al., 2007, Vomund et al., 2013, Fekete et al., 2017, Rosin and Kurrasch, 2018). These investigations have reported good success in terms of both embryo survival rates and the effectiveness of gene transfer as revealed by measurements after birth and into adolescence. To our knowledge, there are only a few in utero ICV injection studies in rodent models that have administered non-genetic material without electroporation directly into the embryonic brain for purposes of providing pharmacological manipulations. In two such reports, direct injections of neuroimmune signaling molecules, the growth factor TGF-B1 that promotes radial glia differentiation (Stipursky et al., 2014) and maternal autoantibodies related to autism spectrum disorders (Camacho et al., 2014), have been performed, with little complication and significant success in demonstrating subsequent changes in target molecules and target regions.
In this manuscript, we use this technique of in utero ICV injection to accomplish two goals. The first is to determine if this technique is a viable method for investigating and directly manipulating mechanisms in the embryonic brain. The second is to test whether the effects in the offspring produced by maternal manipulations of a neuroimmune agent during pregnancy can be reproduced by delivering this agent into the embryonic brain, indicating that they reflect direct actions on the embryo rather than indirect consequences from changes in the mother. Specifically, using the in utero ICV injection technique, we delivered CCL2, a potent signaling molecule, directly into the third ventricle of the embryo at E14, when hypothalamic neurogenesis peaks (Ifft, 1972), and investigated in the embryo just before birth at E19 its effects on neuroimmune function in both the highly proliferative NEP surrounding the third ventricle and the more distant neuronal systems in the LH. In the NEP, we examined the CCL2-containing radial glia progenitor cells in addition to their processes in the medial hypothalamus (mHYP) that facilitate neuronal migration and then tested the CCL2 and MCH neurons in the LH, the site of their final destination. The results obtained with embryonic ICV CCL2 injection reveal very similar effects on the embryo as those produced by maternal peripheral administration of CCL2. In addition to validating this technique which negates potential confounding effects of maternal manipulations that indirectly alter the embryonic and amniotic environment, these results provide strong evidence for the involvement of a localized CCL2 system in the embryo brain in mediating the effects of maternal manipulations. They specifically demonstrate how CCL2 functions in an autoregulatory and sexually dimorphic manner to stimulate the radial glia progenitor cells and processes in which it colocalizes and the development of CCL2 neurons in the LH along with MCH neurons which themselves grow to colocalize CCL2 as well as CCR2.
Section snippets
Experimental procedures
All procedures were conducted in a fully accredited AAALAC facility (22 °C, 12:12-h light–dark cycle with lights off at 8 am), in accordance with protocols approved by The Rockefeller University Animal Care and Use Committee and consistent with the NIH Guide to the Care and Use of Laboratory Animals.
Technique involving ICV injection of CCL2 at E14 and brain analyses at E19
To more fully understand the technique of in utero ICV injection, we used a dye solution (0.1% Fast Green in normal saline) to reveal the nature and extent of its spread and performed histological analyses to verify the site of the injection and assess the extent of damage. As illustrated in the photomicrograph of an E16 embryo sacrificed 2 h after injection (Fig. 2A), the injection through the anterior fissure of the dye solution (1 µl) shows from the surface of the skull how the dye is almost
Discussion
Studies investigating prenatal exposure to teratogenic compounds are generally limited to maternal peripheral manipulations, precluding an analysis of disturbances that reflect actions directly within the fetal brain. Such functional clarity at the fetal level, however, is crucial for a comprehensive understanding of embryonic mechanisms mediating the effects observed subsequently in the offspring, as well as for developing effective therapeutic interventions. The current study demonstrates the
Acknowledgements
This research was supported by National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under Award Number R01AA024798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors would like to thank Ms. Gazal Gulati for her assistance with the literature, references and figures. We also extend gratitude to The Rockefeller University’s Bio-Imaging Resource Center and Translational Technology
Declaration of interest
None.
References (76)
- et al.
Gender differences in alcohol-induced neurotoxicity and brain damage
Toxicology
(2013) - et al.
Radial glial cells: key organisers in CNS development
Int J Biochem Cell Biol
(2014) - et al.
Perturbation of chemokine networks by gene deletion alters the reinforcing actions of ethanol
Behav Brain Res
(2005) - et al.
Embryonic intraventricular exposure to autism-specific maternal autoantibodies produces alterations in autistic-like stereotypical behaviors in offspring mice
Behav Brain Res
(2014) - et al.
Prenatal exposure to ethanol stimulates hypothalamic ccr2 chemokine receptor system: Possible relation to increased density of orexigenic peptide neurons and ethanol drinking in adolescent offspring
Neuroscience
(2015) - et al.
CCL2/CCR2 chemokine system in embryonic hypothalamus: involvement in sexually dimorphic stimulatory effects of prenatal ethanol exposure on peptide-expressing neurons
Neuroscience
(2020) - et al.
CCL2/CCR2 system in neuroepithelial radial glia progenitor cells: Involvement in stimulatory, sexually dimorphic effects of maternal ethanol on embryonic development of hypothalamic peptide neurons
Neuroscience
(2012) - et al.
Neuroimmune basis of alcoholic brain damage
Int Rev Neurobiol
(2014) - et al.
Neuroimmune mechanisms of alcohol and drug addiction
Int Rev Neurobiol
(2014) - et al.
The beta-chemokines ccl2 and ccl7 are two novel differentiation factors for midbrain dopaminergic precursors and neurons
Exp Cell Res
(2008)