Brain-wide genetic mapping identifies the indusium griseum as a prenatal target of pharmacologically unrelated psychostimulants Janos Fuzika,b, Sabah Rehmana,1, Fatima Giracha,1, Andras G. Miklosia,1, Solomiia Korchynskaa, Gloria Arquea, Roman A. Romanova, János Hanicsc,d, Ludwig Wagnere, Konstantinos Meletisb, Yuchio Yanagawaf, Gabor G. Kovacsg, Alán Alpárc,d, Tomas G. M. Hökfeltb,2, and Tibor Harkanya,b,2 aDepartment of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, A-1090 Vienna, Austria; bDepartment of Neuroscience, Biomedicum, Karolinska Institutet, SE-17165 Stockholm, Sweden; cSE NAP Research Group of Experimental Neuroanatomy and Developmental Biology, Semmelweis University, H-1085 Budapest, Hungary; dDepartment of Anatomy, Histology, and Embryology, Semmelweis University, H-1085 Budapest, Hungary; eUniversity Clinic for Internal Medicine III, General Hospital Vienna, A-1090 Vienna, Austria; fDepartment of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511 Japan; and gNeurodegeneration Research Group, Institute of Neurology, Medical University of Vienna, A-1090 Vienna, Austria Contributed by Tomas G. M. Hökfelt, October 10, 2019 (sent for review March 11, 2019; reviewed by Antonello Bonci, Beat Schwaller, and Carsten T. Wotjak) Psychostimulant use is an ever-increasing socioeconomic burden, circuitry to trigger addiction (2). Therefore, any brain region that including a dramatic rise during pregnancy. Nevertheless, brain-wide receives significant dopamine input is at risk upon excess exposure effects of psychostimulant exposure are incompletely understood. to psychoactive substances. Here, we performed Fos-CreERT2–based activity mapping, correlated The consumption of illicit or legal drugs during pregnancy is a for pregnant mouse dams and their fetuses with amphetamine, primary health concern. Besides counteracting medical condi- nicotine, and caffeine applied acutely during midgestation. While tions, such as hyperemesis gravidarum by cannabis and canna- light-sheet microscopy-assisted intact tissue imaging revealed drug- binoids (8), the use of psychostimulants is reasoned by improving and age-specific neuronal activation, the indusium griseum (IG) forgetfulness, mood, and stress. Nevertheless, the efficient cross- + appeared indiscriminately affected. By using GAD67gfp/ mice placental transfer of these structurally distinct compounds (9–11) we subdivided the IG into a dorsolateral domain populated by prompts caution for their direct, indiscriminate, and likely γ-aminobutyric acidergic interneurons and a ventromedial segment adverse effect, particularly if used repeatedly. Mechanistically, containing glutamatergic neurons, many showing drug-induced dopaminergic neurons and their long-range projections to the activation and sequentially expressing Pou3f3/Brn1 and secretagogin forebrain form during the first trimester of human brain devel- (Scgn) during differentiation. We then combined Patch-seq and circuit opment (12), thereby being poised to modulate the migration mapping to show that the ventromedial IG is a quasi-continuum of + and neuritogenesis of telencephalic neurons over long periods glutamatergic neurons (IG-Vglut1 ) reminiscent of dentate granule (13, 14). Nevertheless, the brain-wide distribution of fetal neurons cells in both rodents and humans, whose dendrites emanate perpen- dicularly toward while their axons course parallel with the superior + + Significance longitudinal fissure. IG-Vglut1 neurons receive VGLUT1 and + VGLUT2 excitatory afferents that topologically segregate along their somatodendritic axis. In turn, their efferents terminate in the Drug abuse during pregnancy carries significant risk for im- olfactory bulb, thus being integral to a multisynaptic circuit that paired fetal development. Here, we show that episodic expo- could feed information antiparallel to the olfactory–cortical path- sure to amphetamine, nicotine, or caffeine during pregnancy way. In IG-Vglut1+ neurons, prenatal psychostimulant exposure induces various patterns of neuronal activity in the fetal brain. delayed the onset of Scgn expression. Genetic ablation of Scgn was The indusium griseum, situated parallel to the central surface then found to sensitize adult mice toward methamphetamine-induced of the cortical hemispheres, is indiscriminately activated. epilepsy. Overall, our study identifies brain-wide targets of the Herein, psychostimulants preferentially target glutamatergic + + neurons and delay their differentiation. Particularly, expression most common psychostimulants, among which Scgn /Vglut1 2+ neurons of the IG link limbic and olfactory circuits. of secretagogin, a Ca -sensor protein, is deregulated, which is significant because its loss impairs the integration of information that flows along limbic and olfactory circuits. Cumulatively, we amphetamine | caffeine | human | nicotine | Patch-seq describe psychostimulant-sensitive brain regions and unique neuronal subtypes whose developmental delay disrupts circuit rug abuse is a major socioeconomic burden which affects wiring such that behavioral abnormalities can manifest in off- Dmany age groups and both genders (1, 2). Psychostimulants spring prenatally exposed to psychostimulants. areamongthemostwidelyuseddrugs,andbesidesillicitsub- stances like amphetamine and its derivatives include caffeine Author contributions: T.G.M.H. and T.H. designed research; J.F., S.R., F.G., A.G.M., S.K., (coffee) and nicotine (tobacco). Molecular and structural deter- G.A., R.A.R., J.H., and A.A. performed research; L.W., Y.Y., and G.G.K. contributed new minants mediating the action of these drugs on neurons differ reagents/analytic tools; J.F., S.R., F.G., A.G.M., S.K., G.A., R.A.R., J.H., K.M., G.G.K., A.A., and T.H. analyzed data; and J.F., T.G.M.H., and T.H. wrote the paper. fundamentally. Amphetamine is excitatory by increasing extracel- Reviewers: A.B., Global Institutes on Addiction; B.S., University of Fribourg; and C.T.W., lular dopamine levels through inhibition (or even reversal) of Max Planck Institute of Psychiatry. dopamine transport for cellular reuptake (1, 3, 4). Caffeine an- The authors declare no competing interest. tagonism at the adenosine A2A receptor (5) (and less so at A1 and Published under the PNAS license. A3) is a catalyst for catecholamine (particularly dopamine) and 1S.R., F.G., and A.G.M. contributed equally to this work. acetylcholine release (6). Nicotine activates ionotropic nicotinic 2To whom correspondence may be addressed. Email: [email protected] or tibor. acetylcholine receptors to stimulate synaptic neurotransmission, [email protected]. particularly in the dopaminergic reward circuitry (7). Despite dif- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ferences in their mechanism of action, a common denominator of doi:10.1073/pnas.1904006116/-/DCSupplemental. psychostimulant action is their ability to prime the brain’s reward First published December 3, 2019. 25958–25967 | PNAS | December 17, 2019 | vol. 116 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1904006116 Downloaded by guest on September 26, 2021 that are particularly sensitive to psychostimulants and differences can express c-Fos in response to glutamate and inflammatory between neural foci of fetal vs. maternal drug sensitivity remain stimuli (26, 33). The preferential labeling of neurons in the fetal ambiguous. brain is also compatible with the late onset of astrogliogenesis, Here, we used immediate early gene-based mapping of tran- peaking neonatally, in rodents (34). + sient cellular activity (15) to mark cell contingents (particularly ZsGreen1 activity mapping in serial coronal sections of the neurons and endothelial cells) that were activated by amphet- fetal forebrain from saline-injected dams at E17.5 (Fig. 1F and SI amine, nicotine, or caffeine during the period when neurogenesis Appendix, Figs. S2 and S3) revealed basal Fos-CreERT2 activity peaks in the mouse prosencephalon (that is, embryonic days [E] in motor and sensory cortices, hippocampus proper, and supra- 14 to 16) (16, 17) and reconstructed their distribution by quan- chiasmatic nucleus (SCN) of the hypothalamus. Considering that titative light-sheet microscopy (18). Besides finding critical dif- pacemaker systems of the SCN, including both neurons and ferences in the regional density of genetically tagged cell astroglia (33, 35), can diurnally up-regulate Fos messenger RNA contingents, the medial preoptic area (MPN), arcuate nucleus (mRNA) expression, we interpreted ZsGreen1 labeling in this (ARC) of the hypothalamus, and the indusium griseum (IG), all hypothalamic area as an innate biological control, particularly receiving dense dopamine afferentation or containing dopami- because of the uniform (treatment-independent) size of the + nergic neurons themselves (19), emerged as invariably affected ZsGreen1 cell cohort (suggesting a ceiling effect due to rhyth- fetal brain regions. This genetic model also revealed substantial mic transcriptional activity; Fig. 1G and SI Appendix, Figs. S3 differences in brain-wide cell activation patterns between dams and S4). All drugs induced ZsGreen1 expression in the ventral and their fetuses. We have then conceptually linked previous tegmental area (SI Appendix, Fig. S2), which we have taken as (20) and present findings on neuronal heterogeneity of the IG to another positive control, showing the drug-induced activation of show that glutamatergic