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Intermediate lateral lemniscus (ILL) and Locus coeruleus (LC) nuclei are present in the absence of Shh. a–h Transversal sections to r1 at wild-type (a, e) and mutant (b, f) E14.5 and wild-type (c, g) and mutant (d, h) E18.5 immunoreacted against PAX7 (a, b), double immunoreacted against Pax7 (brown) and POU4F1 (black c, d) and double reacted against TH (brown) and GAD67 (black e–h). Some Pax7-positive neurons migrate radially towards the pial surface,
Source publication
Hindbrain rhombomeres in general are differentially specified molecularly by unique combinations of Hox genes with other developmental genes. Rhombomere 1 displays special features, including absence of Hox gene expression. It lies within the hindbrain range of the Engrailed genes (En1, En2), controlled by the isthmic organizer via diffusion of FGF...
Contexts in source publication
Context 1
... TH-or Pax7-expressing populations likewise originated in the alar plate that normally migrate selectively into the lateral part of the basal plate; these populations include the noradrenergic locus coeruleus (LC) at periventricular levels and an intermediate part of the lateral lemniscal and paralemniscal populations, superfi- cially (ILL, IPL; Fig. 6a, c). These nuclei seemed largely unaffected by the absence of Shh function. The Pax7- positive neurons that originate the ILL share their origin with the IP and medial tegmental populations described above. Jointly with them, they first migrate tangentially out of the Pax7-positive ventral alar plate progenitor domain into the ...
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... tangentially out of the Pax7-positive ventral alar plate progenitor domain into the periventricular stratum of the lateral basal plate, as seen at E12.5 (not shown). By E13.5 the ILL/IPL cells start to migrate radially within the lateral basal plate, instead of approaching the midline (Fig. 5g, j). At E14.5 they have reached the pial surface (Fig. 6a, b). Nevertheless, these ILL and IPL cell populations seemed more dispersed at E18.5 in conditional mutant mice than in wild-type speci- mens, suggesting some final disorganization, possibly due to background abnormality of the basal plate under experimental conditions (Fig. 6c, ...
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... the midline (Fig. 5g, j). At E14.5 they have reached the pial surface (Fig. 6a, b). Nevertheless, these ILL and IPL cell populations seemed more dispersed at E18.5 in conditional mutant mice than in wild-type speci- mens, suggesting some final disorganization, possibly due to background abnormality of the basal plate under experimental conditions (Fig. 6c, ...
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... neurons that constitute the LC are strongly immu- noreactive for tyrosine hydroxylase and were clearly visi- ble at the periventricular stratum of the lateral basal plate at E14.5 (Fig. 6e, f). The LC seemed unaltered in the con- ditional mutant at E18.5; it occupied a position close to the ventricle, as in the wild-type (Fig. 6g, h). TH immunore- action also showed that the substantia nigra (SN) is severely diminished in the midbrain, though a remnant persists, as has been already described previously (Fig. 6g, h; ...
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... neurons that constitute the LC are strongly immu- noreactive for tyrosine hydroxylase and were clearly visi- ble at the periventricular stratum of the lateral basal plate at E14.5 (Fig. 6e, f). The LC seemed unaltered in the con- ditional mutant at E18.5; it occupied a position close to the ventricle, as in the wild-type (Fig. 6g, h). TH immunore- action also showed that the substantia nigra (SN) is severely diminished in the midbrain, though a remnant persists, as has been already described previously (Fig. 6g, h; Perez-Balaguer et al. ...
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... basal plate at E14.5 (Fig. 6e, f). The LC seemed unaltered in the con- ditional mutant at E18.5; it occupied a position close to the ventricle, as in the wild-type (Fig. 6g, h). TH immunore- action also showed that the substantia nigra (SN) is severely diminished in the midbrain, though a remnant persists, as has been already described previously (Fig. 6g, h; Perez-Balaguer et al. ...
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... d Sagittal sections of a wild-type (c) and mutant (d) E15.5 mouse brain immunoreacted with antibodies against a-5HT. e-h and k-p Selected serial horizontal sections through the hindbrain in wild- type (e) and mutant (f) E15.5 and wild-type (g, k, m, o) and mutant (h, l, n, p) E12.5 mouse brains immunoreacted with antibodies against a-5HT (e, f), a- NKX2.2 (g, h), a-ISLET1 (k, m, l, n) and a- NKX6.1 (o, p). The dotted lines in a and c indicate the section plane represented in (e, f) and (g, h), respectively. ...
Citations
... Moreover, with NicheCompass we were able to identify the Floor Plate as another CNS-associated niche. This niche was enriched in the Shh combined interaction GP, consistent with the ligand Shh being secreted from the floor plate 86 . Accordingly, we found specific expression of bona-fide floor plate marker genes in this niche (Supplementary Fig. 5). ...
Spatial omics holds great potential to elucidate tissue architecture by dissecting underlying cell niches and cellular interactions. However, we lack an end-to-end computational framework that can effectively integrate different spatial omics tissue samples, quantitatively characterize cell niches based on biological knowledge of cell-cell communication and transcriptional regulation pathways, and discover spatial molecular programs of cells. We present NicheCompass, a graph deep learning method designed based on the principles of cellular communication. It utilizes existing knowledge of inter- and intracellular interaction pathways to learn an interpretable latent space of cells across multiple tissue samples, enabling the construction and querying of spatial reference atlases. NicheCompass learns the activity of an interaction pathway by modeling the process through the lens of cells receiving and processing signals from their tissue microenvironment, using a variety of mechanisms involving metabolic interactions, ligand-receptor interactions including downstream regulation, and regulons. In addition to leveraging existing knowledge, NicheCompass can learn novel spatially variable gene programs to model variation in the tissue. We showcase a comprehensive workflow encompassing data integration, niche identification, and functional interpretation, and demonstrate that NicheCompass outperforms existing approaches. NicheCompass is broadly applicable to spatial transcriptomics data, which we illustrate by mapping the architecture of diverse tissues during mouse embryonic development, and delineating basal (KRT14) and luminal (KRT8) tumor cell niches in human breast cancer. Moreover, we introduce fine-tuning-based spatial reference mapping, revealing an SPP1+ macrophage-dominated tumor microenvironment in non-small cell lung cancer patients. We further extend NicheCompass to multimodal spatial profiling of gene expression and chromatin accessibility, identifying distinct white matter niches in the mouse brain. Finally, we apply NicheCompass to a whole mouse brain spatial atlas with 8.4 million cells across 239 tissue sections from four mice, demonstrating its ability to build foundational, interpretable spatial representations for entire organs. Overall, NicheCompass provides a novel end-to-end workflow for analyzing large-scale spatial omics data.
... Immunocytochemistry in paraffin-embedded sections. The brains were paraffin-embedded and 10 µm sections were cut in the sagittal or coronal plane, serially mounted on glass slides, and immunostained as performed previously (Moreno-Bravo et al., 2014). The images were collected using a LEICA MZ16FA stereomicroscope equipped with a Leica DFC550 digital camera. ...
Gestational exposure of mice to valproic acid (VPA) is one currently used experimental model for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). In the present study we hypothesized that the reduction of dopaminergic source neurons of the VTA, followed by perturbed growth of the mesotelencephalic dopamine pathway (MT), should also modify pattern formation in the dopaminoceptive target regions (particularly its mesoaccumbens/mesolimbic portion). Here, we investigated VPA-evoked cellular morphological (apoptosis-frequency detected by Caspase-3, abundance of Ca-binding proteins, CaBP), as well as synaptic proteomic (western blotting) changes, in selected dopaminoceptive subpallial, as compared to pallial, regions of mice, born to mothers treated with 500 mg/kg VPA on day 13.5 of pregnancy. We observed a surge of apoptosis on VPA treatment in nearly all investigated subpallial and pallial regions; with a non-significant trend of similar increase the nucleus accumbens (NAc) at P7, the age at which the MT pathway reduction has been reported (also supplemented by current findings). Of the CaBPs, calretinin (CR) expression was decreased in pallial regions, most prominently in retrosplenial cortex, but not in the subpallium of P7 mice. Calbindin-D 28K (CB) was selectively reduced in the caudate-putamen (CPu) of VPA exposed animals at P7 but no longer at P60, pointing to a potency of repairment. The VPA-associated overall increase in apoptosis at P7 did not correlate with the abundance and distribution of CaBPs, except in CPu, in which the marked drop of CB was negatively correlated with increased apoptosis. Abundance of parvalbumin (PV) at P60 showed no significant response to VPA treatment in any of the observed regions we did not find colocalization of apoptotic (Casp3+) cells with CaBP-immunoreactive neurons. The proteomic findings suggest reduction of tyrosine hydroxylase in the crude synaptosome fraction of NAc, but not in the CPu, without simultaneous decrease of the synaptic protein, synaptophysin, indicating selective impairment of dopaminergic synapses. The morpho-functional changes found in forebrain regions of VPA-exposed mice may signify dendritic and synaptic reorganization in dopaminergic target regions, with potential translational value to similar impairments in the pathogenesis of human ASD.
... As the main cholinergic system in the rostral hindbrain [10], pontine-tegmental cholinergic neurons (PTCNs) send abundant axons along three major trajectories and are involved in various functions [11]. In the ascending dorsal circuits, PTCNs mediate prefrontal serotonin release from the DR [12] and participate in multiple functions, including auditory sensation [13], sensorimotor function [14], and spatial memory [15], by targeting different thalamic nuclei. ...
Background
Based on their anatomical location, rostral projections of nuclei are classified as ascending circuits, while caudal projections are classified as descending circuits. Upper brainstem neurons participate in complex information processing and specific sub-populations preferentially project to participating ascending or descending circuits. Cholinergic neurons in the upper brainstem have extensive collateralizations in both ascending and descending circuits; however, their single-cell projection patterns remain unclear because of the lack of comprehensive characterization of individual neurons.
Results
By combining fluorescent micro-optical sectional tomography with sparse labeling, we acquired a high-resolution whole-brain dataset of pontine-tegmental cholinergic neurons (PTCNs) and reconstructed their detailed morphology using semi-automatic reconstruction methods. As the main source of acetylcholine in some subcortical areas, individual PTCNs had abundant axons with lengths up to 60 cm and 5000 terminals and innervated multiple brain regions from the spinal cord to the cortex in both hemispheres. Based on various collaterals in the ascending and descending circuits, individual PTCNs were grouped into four subtypes. The morphology of cholinergic neurons in the pedunculopontine nucleus was more divergent, whereas the laterodorsal tegmental nucleus neurons contained richer axonal branches and dendrites. In the ascending circuits, individual PTCNs innervated the thalamus in three different patterns and projected to the cortex via two separate pathways. Moreover, PTCNs targeting the ventral tegmental area and substantia nigra had abundant collaterals in the pontine reticular nuclei, and these two circuits contributed oppositely to locomotion.
Conclusions
Our results suggest that individual PTCNs have abundant axons, and most project to various collaterals in the ascending and descending circuits simultaneously. They target regions with multiple patterns, such as the thalamus and cortex. These results provide a detailed organizational characterization of cholinergic neurons to understand the connexional logic of the upper brainstem.
... In fact, DLL and ILL misidentification has occurred several times in the literature and indeed, Math1 (MacHold and Fishell, 2005), Wnt3a (Louvi et al., 2007), and Slc17a6 (VGluT2) (Rose et al., 2009;Di Bonito et al., 2013) labeling in the ILL was misinterpreted as the DLL as noted by Di . Similarly, the Pax7 labeling indicated in the ILL (Moreno-Bravo et al., 2014) corresponds rather to the DLL, at least in the adult brain (present work). The Hcn channel family (Hcn1-Hcn4) participates in the excitability of cells in the auditory system (He et al., 2014). ...
IntroductionThe dorsal (DLL), intermediate (ILL), and ventral (VLL) lateral lemniscus nuclei are relay centers in the central auditory pathway of the brainstem, commonly referred to as the lateral lemniscus nuclei (LLN). The LLN are situated in the prepontine and pontine hindbrain, from rhombomeres 1 to 4, extending from the more rostral DLL to the caudal VLL, with the ILL lying in between. These nuclei can be distinguished morphologically and by topological and connectivity criteria, and here, we set out to further characterize the molecular nature of each LLN.Methods
We searched in situ hybridization studies in the Allen Mouse Brain Atlas for genes differentially expressed along the rostrocaudal axis of the brainstem, identifying 36 genes from diverse functional families expressed in the LLN.ResultsAvailable information in the databases indicated that 7 of these 36 genes are either associated with or potentially related to hearing disorders.DiscussionIn conclusion, the LLN are characterized by specific molecular profiles that reflect their rostrocaudal organization into the three constituent nuclei. This molecular regionalization may be involved in the etiology of some hearing disorders, in accordance with previous functional studies of these genes.
... The specification of the different dA/dB subclasses requires coordinated signaling cues that arise from the roof and floor plate (FP) (i.e., BMP, Wnt, and SHH), and provide positional information which leads to the birth of individual neuronal fates (Liem et al., 1997;Lee et al., 1998Lee et al., , 2000Briscoe et al., 1999;Gaufo et al., 2000;Vogel-Höpker and Rohrer, 2002;Müller et al., 2005;Storm et al., 2009;Tilleman et al., 2010;Moreno-Bravo et al., 2014;Lihua et al., 2019). The mechanisms by which these morphogens act to pattern hindbrain dINs are not fully understood and will not be discussed further in this review. ...
Unraveling the inner workings of neural circuits entails understanding the cellular origin and axonal pathfinding of various neuronal groups during development. In the embryonic hindbrain, different subtypes of dorsal interneurons (dINs) evolve along the dorsal-ventral (DV) axis of rhombomeres and are imperative for the assembly of central brainstem circuits. dINs are divided into two classes, class A and class B, each containing four neuronal subgroups (dA1-4 and dB1-4) that are born in well-defined DV positions. While all interneurons belonging to class A express the transcription factor Olig3 and become excitatory, all class B interneurons express the transcription factor Lbx1 but are diverse in their excitatory or inhibitory fate. Moreover, within every class, each interneuron subtype displays its own specification genes and axonal projection patterns which are required to govern the stage-by-stage assembly of their connectivity toward their target sites. Remarkably, despite the similar genetic landmark of each dINs subgroup along the anterior-posterior (AP) axis of the hindbrain, genetic fate maps of some dA/dB neuronal subtypes uncovered their contribution to different nuclei centers in relation to their rhombomeric origin. Thus, DV and AP positional information has to be orchestrated in each dA/dB subpopulation to form distinct neuronal circuits in the hindbrain. Over the span of several decades, different axonal routes have been well-documented to dynamically emerge and grow throughout the hindbrain DV and AP positions. Yet, the genetic link between these distinct axonal bundles and their neuronal origin is not fully clear. In this study, we reviewed the available data regarding the association between the specification of early-born dorsal interneuron subpopulations in the hindbrain and their axonal circuitry development and fate, as well as the present existing knowledge on molecular effectors underlying the process of axonal growth.
... 268020234), αNTN1 (1:500; RD Systems MAB1109), and αROBO3 (kindly provided by Dr. Fujio Murakami, Osaka University, and 1:300; AF3076/R&D Systems). In both cases, the protocols were performed as previously described (Moreno-Bravo et al., 2014). ...
... The Open Neural Tubes (ONTs) were performed as described previously by Moreno-Bravo et al. (2014). Briefly, neural tubes of E12.5 or E13.5 embryos were dissected and opened along the dorsal midline, and the telencephalic vesicles and hypothalamus FIGURE 1 | Longitudinal navigation of habenular axons through the SNc and VTA. ...
... We next focused on demonstrating the possible attractive role of SNc and VTA on mHb axons. We developed an in vitro assay where SNc explants from a GFP +/− mouse brain were ectopically placed in an organotypic neural tube culture (ONTs, Moreno-Bravo et al., 2014). As control, we used p1 alar plate explants ( Figure 3A). ...
The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.
... Because of their emphasis on late developmental gene markers, these authors did notice the postulated VPM migration Nouri and Awatramani, 2017), nor compared VPM markers with those of the RM area. A convergent migration of molecularly differentially defined neuronal types into a single adult cell nucleus has been described before for the avian and mouse interpeduncular nucleus (Lorente-Cánovas et al., 2012;Moreno-Bravo et al., 2014;Ruiz-Reig et al., 2017;García-Guillén et al., 2020). ...
The tuberal hypothalamic ventral premamillary nucleus (VPM) described in mammals links olfactory and metabolic cues with mating behavior and is involved in the onset of puberty. We offer here descriptive and experimental evidence on a migratory phase in the development of this structure in mice at E12.5–E13.5. Its cells originate at the retromamillary area (RM) and then migrate tangentially rostralward, eschewing the mamillary body, and crossing the molecularly distinct perimamillary band, until they reach a definitive relatively superficial ventral tuberal location. Corroborating recent transcriptomic studies reporting a variety of adult glutamatergic cell types in the VPM, and different projections in the adult, we found that part of this population heterogeneity emerges already early in development, during tangential migration, in the form of differential gene expression properties of at least 2–3 mixed populations possibly derived from subtly different parts of the RM. These partly distribute differentially in the core and shell parts of the final VPM. Since there is a neighboring acroterminal source of Fgf8, and Fgfr2 is expressed at the early RM, we evaluated a possible influence of Fgf8 signal on VPM development using hypomorphic Fgf8 neo/null embryos. These results suggested a trophic role of Fgf8 on RM and all cells migrating tangentially out of this area (VPM and the subthalamic nucleus), leading in hypomorphs to reduced cellularity after E15.5 without alteration of the migrations proper.
... Both the IPR and IPC are further divided along the mediolateral and dorsoventral axes into several sub-nuclei (Lorente-Cánovas et al., 2012). Subsequently, it was shown that this a nucleus follows a similar developmental course in mice (Moreno-Bravo et al., 2014;Ruiz-Reig et al., 2019;García-Guillén et al., 2020). ...
... The 135 genes were regionally expressed in the developing IPN, i.e.: they were expressed in at least one of the principal domains of the IPN (Pro, IPR, IPC). This segmentation of the IPN was initially proposed in the chick (Lorente-Cánovas et al., 2012) and later confirmed in the mouse (Moreno-Bravo et al., 2014;Ruiz-Reig et al., 2019;García-Guillén et al., 2020). Here, we further characterized the molecular profiles for each anteroposterior segment of the IPN located in their respective neuromeric units (Ist, r1-r and r1-c: Aroca and Puelles, 2005;Alonso et al., 2013). ...
... In addition, we also describe the molecular regionalization of the deep to superficial areas of the IPR (IPRa, IPRi, IPRb). To date, 5 TFs have been used as markers for diverse IPN populations: Nkx6.1, Pax7, Otp, Otx2, and Irx2 (Lorente-Cánovas et al., 2012;Moreno-Bravo et al., 2014;García-Guillén et al., 2020). Remarkably, our work identified up to 50 transcriptional regulators within the IPN, as well as other genes involved in different aspects of neural development. ...
The interpeduncular nucleus (IPN) is a highly conserved limbic structure in the vertebrate brain, located in the isthmus and rhombomere 1. It is formed by various populations that migrate from different sites to the distinct domains within the IPN: the prodromal, rostral interpeduncular, and caudal interpeduncular nuclei. The aim here was to identify genes that are differentially expressed across these domains, characterizing their putative functional roles and interactions. To this end, we screened the 2,038 genes in the Allen Developing Mouse Brain Atlas database expressed at E18.5 and we identified 135 genes expressed within the IPN. The functional analysis of these genes highlighted an overrepresentation of gene families related to neuron development, cell morphogenesis and axon guidance. The interactome analysis within each IPN domain yielded specific networks that mainly involve members of the ephrin/Eph and Cadherin families, transcription factors and molecules related to synaptic neurotransmission. These results bring to light specific mechanisms that might participate in the formation, molecular regionalization, axon guidance and connectivity of the different IPN domains. This genoarchitectonic model of the IPN enables data on gene expression and interactions to be integrated and interpreted, providing a basis for the further study of the connectivity and function of this poorly understood nuclear complex under both normal and pathological conditions.
... Neuronal migration is an important feature of brain development, in some cases involving the displacement of considerable cell populations toward different coordinates. In addition to the classic examples of migration, such as that in the precerebellar nuclei (Bloch-Gallego et al., 2005), recent data has shown that complex migratory events contribute to the formation of the interpeduncular nucleus (IPN) (Lorente-Cánovas et al., 2012;Moreno-Bravo et al., 2014;Ruiz-Reig et al., 2019). In vertebrates, the IPN is a complex hindbrain structure and it is the principal target of the retroflex tract that comes from the medial habenula (mHb) (Herkenham and Nauta, 1979;Contestabile and Flumerfelt, 1981). ...
... In behavioral and functional studies, the mHb-IPN pathway has been implicated in learning and memory, affective states and mood-related psychiatric conditions (Klemm, 2004;Hikosaka, 2010). However, the development of the IPN has only recently been studied, in particular in the chick (Lorente-Cánovas et al., 2012) and mouse (Moreno-Bravo et al., 2014;Ruiz-Reig et al., 2019). ...
... Our previous experimental study in the chick demonstrated that the IPN is formed in the rostral (prepontine) hindbrain, containing cells from both alar and basal populations (Lorente-Cánovas et al., 2012). Each of the IPN populations follows a specific dorsoventral migratory trajectory from its original periventricular position, ultimately converging at a more medial site and settling down in a sub-pial location across the median floor plate of the isthmus (Ist) and hindbrain rhombomere 1 (r1) (Lorente-Cánovas et al., 2012;Moreno-Bravo et al., 2014). At the end of this process, the so-called rostral (IPR) and caudal (IPC) domains of the IPN lie in the rostral and caudal parts of r1 (r1-r, r1-c), respectively, while the smaller rostral prodromal (Pro) domain is located in the isthmic region. ...
The interpeduncular nucleus (IPN) is a hindbrain structure formed by three main subdivisions, the prodromal (Pro) domain located at the isthmus (Ist), and the rostral and caudal interpeduncular domains (IPR, IPC) within rhombomere 1 (r1). Various cell populations can be detected in the IPN through the expression of the Nkx6.1, Otp, Otx2, Pax7, and/or Irx2 transcription factors. These cell populations follow independent dorsoventral tangential and radial migratory routes targeting the ventral paramedian region of Ist and r1. Here we set out to examine the influence of the Netrin-1/DCC pathway on these migrations, since it is known to regulate other processes of neuronal migration in the brain. To this end, we analyzed IPN development in late gestational wild-type and DCC–/– mice, using mainly in situ hybridization (ISH) to identify the cells expressing each of the aforementioned genes. We found that the migration of Nkx6.1⁺ and Irx2⁺ cells into the Pro domain was strongly disrupted by the loss of DCC, as occurred with the migration of Pax7⁺, Irx2⁺, and Otp⁺ cells that would normally form the IPR. In addition, there was mild impairment of the migration of the Pax7⁺ and Otx2⁺ cells that form the IPC. These results demonstrate that the Netrin-1/DCC signaling pathway is involved in the migration of most of the IPN populations, mainly affecting those of the Pro and IPR domains of this nucleus. There are psychiatric disorders that involve the medial habenula (mHb)-IPN system, so that this experimental model could provide a basis to study their neurodevelopmental etiology.
... To determine whether VPA treatment affects the general pattern of dopaminergic and non-dopaminergic tracts, we used conventional microscopy on paraffin-embedded sections. The brains were paraffin-embedded and 10 µm sections were cut in the sagittal or coronal plane, serially mounted, and immunohistochemistry was developed as previously described (Moreno-Bravo et al., 2014). The images were collected using a LEICA MZ16FA stereomicroscope equipped with a Leica DFC550 digital camera. ...
Gestational exposure to valproic acid (VPA) is known to cause behavioral deficits of sociability, matching similar alterations in human autism spectrum disorder (ASD). Available data are scarce on the neuromorphological changes in VPA-exposed animals. Here, we focused on alterations of the dopaminergic system, which is implicated in motivation and reward, with relevance to social cohesion. Whole brains from 7-day-old mice born to mothers given a single injection of VPA (400 mg/kg b.wt.) on E13.5 were immunostained against tyrosine hydroxylase (TH). They were scanned using the iDISCO method with a laser light-sheet microscope, and the reconstructed images were analyzed in 3D for quantitative morphometry. A marked reduction of mesotelencephalic (MT) axonal fascicles together with a widening of the MT tract were observed in VPA treated mice, while other major brain tracts appeared anatomically intact. We also found a reduction in the abundance of dopaminergic ventral tegmental (VTA) neurons, accompanied by diminished tissue level of DA in ventrobasal telencephalic regions (including the nucleus accumbens (NAc), olfactory tubercle, BST, substantia innominata). Such a reduction of DA was not observed in the non-limbic caudate-putamen. Conversely, the abundance of TH+ cells in the substantia nigra (SN) was increased, presumably due to a compensatory mechanism or to an altered distribution of TH+ neurons occupying the SN and the VTA. The findings suggest that defasciculation of the MT tract and neuronal loss in VTA, followed by diminished dopaminergic input to the ventrobasal telencephalon at a critical time point of embryonic development (E13-E14) may hinder the patterning of certain brain centers underlying decision making and sociability.
