Abstract: DYT1 dystonia is an inherited disease linked to mutation in the TOR1A gene encoding for the protein torsinA. Although the mechanism by which this genetic alteration leads to dystonia is unclear, multiple lines of clinical evidence suggest a link between dystonia and a reduced dopamine D2 receptor (D2R) availability. Based on this evidence, herein we carried out a comprehensive analysis of electrophysiological, behavioral and signaling correlates of D2R transmission in transgenic mice with the DYT1 dystonia mutation. Electrophysiological recordings from nigral dopaminergic neurons showed a normal responsiveness to D2-autoreceptor function. Conversely, postsynaptic D2R function in hMT mice was impaired, as suggested by the inability of a D2R agonist to re-establish normal corticostriatal synaptic plasticity and supported by the reduced sensitivity to haloperidol-induced catalepsy. Although an in situ hybridization analysis showed normal D1R and D2R mRNA expression levels in the striata of hMT mice, we found a significant decrease of D2R protein, coupled to a reduced ability of D2Rs to activate their cognate Go/i proteins. Of relevance, we found that pharmacological blockade of adenosine A2A receptors (A2ARs) fully restored the impairment of synaptic plasticity observed in hMT mice. Together, our findings demonstrate an important link between torsinA mutation and D2R dysfunction and suggest that A2AR antagonism is able to counteract the deficit in D2R-mediated transmission observed in mutant mice, opening new perspectives for the treatment of this movement disorder.
Abstract: Rhes is a small GTP-binding protein prominently localized in the striatum. Previous findings obtained in cell culture systems demonstrated an involvement of Rhes in cAMP/PKA signalling pathway, at a level proximal to the activation of heterotrimeric G-protein complex. However, its role in the striatum has been, so far, only supposed. Here we studied the involvement of Rhes in dopaminergic signalling, by employing mice with a null mutation in the Rhes gene. We demonstrated that the absence of Rhes modulates cAMP/PKA signalling in both striatopallidal and striatonigral projection neurons by increasing Golf protein levels and, in turn, influencing motor responses challenged by dopaminergic agonist/antagonist. Interestingly, we also show that Rhes is required for a correct dopamine-mediated GTP binding, a function mainly associated to stimulation of dopamine D2 receptors. Altogether, our results indicate that Rhes is an important modulator of dopaminergic transmission in the striatum.
Abstract: The pontine neurons (PN) represent a major source of mossy fiber projections to the cerebellum. During mouse hindbrain development, PN migrate tangentially and sequentially along both the anteroposterior (AP) and dorsoventral (DV) axes. Unlike DV migration, which is controlled by the Netrin-1/Dcc attractive pathway, little is known about the molecular mechanisms guiding PN migration along the AP axis. Here, we show that Hoxa2 and Hoxb2 are required both intrinsically and extrinsically to maintain normal AP migration of subsets of PN, by preventing their premature ventral attraction towards the midline. Moreover, the migration defects observed in Hoxa2 and Hoxb2 mutant mice were phenocopied in compound Robo1;Robo2, Slit1;Slit2, and Robo2;Slit2 knockout animals, indicating that these guidance molecules act downstream of Hox genes to control PN migration. Indeed, using chromatin immunoprecipitation assays, we further demonstrated that Robo2 is a direct target of Hoxa2 in vivo and that maintenance of high Robo and Slit expression levels was impaired in Hoxa2 mutant mice. Lastly, the analysis of Phox2b-deficient mice indicated that the facial motor nucleus is a major Slit signaling source required to prevent premature ventral migration of PN. These findings provide novel insights into the molecular control of neuronal migration from transcription factor to regulation of guidance receptor and ligand expression. Specifically, they address the question of how exposure to multiple guidance cues along the AP and DV axes is regulated at the transcriptional level and in turn translated into stereotyped migratory responses during tangential migration of neurons in the developing mammalian brain.
Abstract: BACKGROUND: Subcortical band heterotopia (SBH, or double cortex syndrome) is a neuronal migration disorder consisting of heterotopic bands of gray matter located between the cortex and the ventricular surface, with or without concomitant pachygyria. Most cases show diffuse or anteriorly predominant (A>P) migration abnormality. All familial and 53% to 84% of sporadic cases with diffuse or A>P SBH harbor a mutation of the DCX gene, leaving the genetic causes unexplained, and genetic counseling problematic, in the remaining patients. Our purpose was to verify the extent to which exonic deletions or duplications of the DCX gene would account for sporadic SBH with A>P gradient but normal gene sequencing. METHODS: We identified 23 patients (22 women, 1 man) with sporadic, diffuse, or anteriorly predominant SBH. After sequencing the DCX gene and finding mutations in 12 (11 women, 1 man), we used multiplex ligation-dependent probe amplification (MLPA) to search for whole-exon deletions or duplications in the 11 remaining women. We used semiquantitative fluorescent multiplex PCR (SQF-PCR) and Southern blot to confirm MLPA findings. RESULTS: MLPA assay uncovered two deletions encompassing exons 3 to 5, and one involving exon 6, in 3 of 11 women (27%) and raised the percentage of DCX mutations from 52% to 65% in our series. SQF-PCR performed in all three women and Southern blot analysis performed in two confirmed the deletions. CONCLUSIONS: MLPA uncovers large genomic deletions of the DCX gene in a subset of patients with SBH in whom no mutations are found after gene sequencing. Deletions of DCX are an underascertained cause of SBH.
Abstract: The Hox code of jawed vertebrates is characterized by the colinear and rostrocaudally nested expression of Hox genes in pharyngeal arches, hindbrain, somites, and limb/fin buds. To gain insights into the evolutionary path leading to the gnathostome Hox code, we have systematically analyzed the expression pattern of the Hox gene complement in an agnathan species, Lethenteron japonicum (Lj). We have isolated 15 LjHox genes and assigned them to paralogue groups (PG) 1-11, based on their deduced amino acid sequences. LjHox expression during development displayed gnathostome-like spatial patterns with respect to the PG numbers. Specifically, lamprey PG1-3 showed homologous expression patterns in the rostral hindbrain and pharyngeal arches to their gnathostome counterparts. Moreover, PG9-11 genes were expressed specifically in the tailbud, implying its posteriorizing activity as those in gnathostomes. We conclude that these gnathostome-like colinear spatial patterns of LjHox gene expression can be regarded as one of the features already established in the common ancestor of living vertebrates. In contrast, we did not find evidence for temporal colinearity in the onset of LjHox expression. The genomic and developmental characteristics of Hox genes from different chordate species are also compared, focusing on evolution of the complex body plan of vertebrates.
Abstract: As a step toward generating a fate map of identified neuron populations in the mammalian hindbrain, we assessed the contributions of individual rhombomeres to the vestibular nuclear complex, a major sensorimotor area that spans the entire rhombencephalon. Transgenic mice harboring either the lacZ or the enhanced green fluorescent protein reporter genes under the transcriptional control of rhombomere-specific Hoxa2 enhancer elements were used to visualize rhombomere-derived domains. We labeled functionally identifiable vestibular projection neuron groups retrogradely with conjugated dextran-amines at successive embryonic stages and obtained developmental fate maps through direct comparison with the rhombomere-derived domains in the same embryos. The fate maps show that each vestibular neuron group derives from a unique rostrocaudal domain that is relatively stable developmentally, suggesting that anteroposterior migration is not a major contributor to the rostrocaudal patterning of the vestibular system. Most of the groups are multisegmental in origin, and each rhombomere is fated to give rise to two or more vestibular projection neuron types, in a complex pattern that is not segmentally iterated. Comparison with studies in the chicken embryo shows that the rostrocaudal patterning of identified vestibular projection neuron groups is generally well conserved between avians and mammalians but that significant species-specific differences exist in the rostrocaudal limits of particular groups. This mammalian hindbrain fate map can be used as the basis for targeting genetic manipulation to specific subpopulations of vestibular projection neurons.
Abstract: BACKGROUND: Little is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of Hoxa2, the only Hox gene expressed up to r2, and of Krox20, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of Hoxa2 by Krox20 in r3. RESULTS: We found that Hoxa2 mutants displayed an impaired oro-buccal reflex, similarly to Krox20 mutants. In contrast, while Krox20 is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency, Hoxa2 inactivation did not affect neonatal breathing frequency. Instead, we found that Hoxa2-/- but not Krox20-/- mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of Hoxa2 expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory. CONCLUSION: Thus, inspiratory shaping and respiratory frequency are under the control of distinct Hox-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.
Abstract: In the mouse trigeminal pathway, sensory inputs from distinct facial structures, such as whiskers or lower jaw and lip, are topographically mapped onto the somatosensory cortex through relay stations in the thalamus and hindbrain. In the developing hindbrain, the mechanisms generating such maps remain elusive. We found that in the principal sensory nucleus, the whisker-related map is contributed by rhombomere 3-derived neurons, whereas the rhombomere 2-derived progeny supply the lower jaw and lip representation. Moreover, early Hoxa2 expression in neuroepithelium prevents the trigeminal nerve from ectopically projecting to the cerebellum, whereas late expression in the principal sensory nucleus promotes selective arborization of whisker-related afferents and topographic connectivity to the thalamus. Hoxa2 inactivation further results in the absence of whisker-related maps in the postnatal brain. Thus, Hoxa2- and rhombomere 3-dependent cues determine the whisker area map and are required for the assembly of the whisker-to-barrel somatosensory circuit.
Abstract: Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.
Abstract: The skeletal structures of the face and throat are derived from cranial neural crest cells (NCCs) that migrate from the embryonic neural tube into a series of branchial arches (BAs). The first arch (BA1) gives rise to the upper and lower jaw cartilages, whereas hyoid structures are generated from the second arch (BA2). The Hox paralogue group 2 (PG2) genes, Hoxa2 and Hoxb2, show distinct roles for hyoid patterning in tetrapods and fishes. In the mouse, Hoxa2 acts as a selector of hyoid identity, while its paralogue Hoxb2 is not required. On the contrary, in zebrafish Hoxa2 and Hoxb2 are functionally redundant for hyoid arch patterning. Here, we show that in Xenopus embryos morpholino-induced functional knockdown of Hoxa2 is sufficient to induce homeotic changes of the second arch cartilage. Moreover, Hoxb2 is downregulated in the BA2 of Xenopus embryos, even though initially expressed in second arch NCCs, similar to mouse and unlike in zebrafish. Finally, Xbap, a gene involved in jaw joint formation, is selectively upregulated in the BA2 of Hoxa2 knocked-down frog embryos, supporting a hyoid to mandibular change of NCC identity. Thus, in Xenopus Hoxa2 does not act redundantly with Hoxb2 for BA2 patterning, similar to mouse and unlike in fish. These data bring novel insights into the regulation of Hox PG2 genes and hyoid patterning in vertebrate evolution and suggest that Hoxa2 function is required at late stages of BA2 development.
Abstract: During development, the vertebrate hindbrain is subdivided along its anteroposterior axis into a series of segmental bulges called rhombomeres. These segments in turn generate a repeated pattern of rhombomere-specific neurons, including reticular and branchiomotor neurons. In amphioxus (Cephalochordata), the sister group of the vertebrates, a bona fide segmented hindbrain is lacking, although the embryonic brain vesicle shows molecular anteroposterior regionalization. Therefore, evaluation of the segmental patterning of the central nervous system of agnathan embryos is relevant to our understanding of the origin of the developmental plan of the vertebrate hindbrain. To investigate the neuronal organization of the hindbrain of the Japanese lamprey, Lethenteron japonicum, we retrogradely labeled the reticulospinal and branchial motoneurons. By combining this analysis with a study of the expression patterns of genes identifying specific rhombomeric territories such as LjKrox20, LjPax6, LjEphC and LjHox3, we found that the reticular neurons in the lamprey hindbrain, including isthmic, bulbar and Mauthner cells, develop in conserved rhombomere-specific positions, similar to those in the zebrafish. By contrast, lamprey trigeminal and facial motor nuclei are not in register with rhombomere boundaries, unlike those of gnathostomes. The trigeminal-facial boundary corresponds to the rostral border of LjHox3 expression in the middle of rhombomere 4. Exogenous application of retinoic acid (RA) induced a rostral shift of both the LjHox3 expression domain and branchiomotor nuclei with no obvious repatterning of rhombomeric segmentation and reticular neurons. Therefore, whereas subtype variations of motoneuron identity along the anteroposterior axis may rely on Hox-dependent positional values, as in gnathostomes, such variations in the lamprey are not constrained by hindbrain segmentation. We hypothesize that the registering of hindbrain segmentation and neuronal patterning may have been acquired through successive and independent stepwise patterning changes during evolution.
Abstract: We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti-cone-opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone-opsins. We named the cloned cone-opsin Ps-RH2. To examine whether brain cone-opsins mediate photic entrainment of circadian locomotor rhythms, we performed post-transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone-opsin Ps-RH2 mRNA in the third cerebral ventricle of pinealectomized-retinectomized lizards previously entrained to a light-dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized-retinectomized lizards to the LD cycle for 6-9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone-opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone-opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.
Abstract: Analysis of mice that carry targeted inactivations of Hox, Nkx and Phox2 homeobox genes revealed their involvement in regional patterning of brain-stem territories, in specification of neuronal identity, in establishment of appropriate patterns of connectivity and in control of neurotransmission. The specific abnormalities generated by such mutations may provide clues to the genetic basis and cellular mechanisms that are involved in human brain-stem developmental disorders.
Abstract: For the past 75 years, vitamin A and its biologically active metabolites, the retinoids, have been the object of intense study in biology and medicine. A large body of evidence demonstrates that these nutrients are essential for normal development and survival of vertebrate embryos, including mammals. In fact, it has been known since the mid-1930s that vitamin A deficiency during pregnancy results in death of the fetus and congenital abnormalities. Similarly, excess dietary intake of vitamin A can also cause teratogenic responses. Among the main targets of both deficiency and excess retinoid-induced teratogenesis are the heart, limbs, craniofacial structures, central nervous system, and the inner ear. Specific malformations are induced in a stage- and dose-dependent manner. Thus, these studies indicate that precise levels and timing of action of vitamin A metabolites are required for normal patterning of embryonic structures. In addition, the discovery of the nuclear receptors for retinoic acid (RA) and other vitamin A derivatives provided a molecular basis to explain how distinct doses of these compounds elicit cell-specific responses via the direct transcriptional activation of a panel of target genes.
Abstract: Little is known about the genetic pathways involved in the early steps of inner ear morphogenesis. Hoxa1 is transiently expressed in the developing hindbrain; its targeted inactivation in mice results in severe abnormalities of the otic capsule and membranous labyrinth. Here we show that a single maternal administration of a low dose of the vitamin A metabolite retinoic acid is sufficient to compensate the requirement for Hoxa1 function. It rescues cochlear and vestibular defects in mutant fetuses without affecting the development of the wildtype fetuses. These results identify a temporal window of susceptibility to retinoids that is critical for mammalian inner ear specification, and provide the first evidence that a subteratogenic dose of vitamin A derivative can be effective in rescuing a congenital defect in the mammalian embryo.
Abstract: Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. Here, we use an inducible system to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. We uncover stage-specific effects of Hoxa2 gain-of-function suggesting a multistep patterning process for hindbrain NC. Most interestingly, we show that Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, normally devoid of Hox expression, towards hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws.
Abstract: The regional and cellular distribution of serotonin type 2C receptor messenger RNA was investigated in autopsy samples of human brain by in situ hybridization histochemistry. The main sites of serotonin receptor type 2C messenger RNA expression were the choroid plexus, cerebral cortex, hippocampus, amygdala, some components of the basal ganglia, the substantia nigra, the substantia innominata and the ventromedial hypothalamus, suggesting that this receptor might be involved in the regulation of different brain functions. Interestingly, in all regions examined, the serotonin type 2C receptor messenger RNA was always restricted to subpopulations of cells, suggesting a specific role, perhaps determined by regionality. A comparison of the in situ hybridization results with those previously obtained by means of radioligand binding experiments suggested that in most of the areas analysed the serotonin type 2C receptors were located at axon terminals.
Abstract: The 5-HT5A receptor is a member of a new subfamily of serotonin [5-hydroxytryptamine (5-HT)] receptors recently cloned from the human and rodent brain. The role of this receptor in normal brain functions as well as its possible involvement in pathological states is still to be determined. We therefore studied the regional distribution and cellular localization of 5-HT5A receptor mRNA in human brain sections from autopsy samples by in situ hybridization histochemistry, in order to obtain anatomical information which might be useful in formulating hypotheses on possible functions subserved by this receptor in the central nervous system (CNS). Our results showed that the main sites of 5-HT5A mRNA expression were the cerebral cortex, hippocampus and cerebellum. In the neocortical regions, the 5-HT5A receptor mRNA was mainly distributed in the layers II-III and V-VI. In the hippocampus, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields expressed 5-HT5A mRNA at high levels. The broad distribution in the neocortex and hippocampus supports the view that the 5-HT5A receptor in these areas might be implicated in high cortical and limbic functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it was highly expressed in the Purkinje cells, in the dentate nucleus and, at a lower level, in the granule cells. Since the cerebellum receives diffuse serotonergic afferents, this finding suggests that the 5-HT5A receptor may have an important role in mediating the effects of 5-HT on cerebellar functions.
Abstract: The distribution of messenger ribonucleic acids (mRNA) for serotonin (5-HT) receptors of 1A, 2A and 1D alpha type (5-HT1A, 5-HT2A, and 5-HT1D alpha) was examined and compared in autoptic human brain by means of in-situ hybridization using cRNA probes, in those areas with the highest density of the receptors, as observed with binding techniques. The results showed that the 5-HT1A receptor mRNA was abundantly expressed in the layers II-VI of all cortical areas under examination, but the highest expression was found in the hippocampus, particularly in the granular cells of the dentate gyrus and in the pyramidal cell layer of the Hammon's horn. The 5-HT2A receptor mRNA was mainly present in the layers III-V of the cortex, with regional differences which were particularly marked in the striate area where the layer IV was specifically labeled. On the other hand, in the hippocampus, 5-HT2A receptor mRNA was restricted to the pyramidal cell layer of the CA1 field of the Hammon's horn. No expression of both 5-HT2A and 5-HT1D alpha receptors was detected in the caudate nucleus and in putamen where only a light labeling by means of the 5-HT1A receptor probe was detected. The 5-HT1D alpha receptor mRNA was found only in the CA3 field of the Hammon's horn. These findings confirm that 5-HT receptors are widely distributed in the brain, but that the different subtypes possess a selective localization in different neuronal populations which, in turn, may express one or more receptors. The regional differences may represent the anatomical substrate of different serotonergic functions and dysfunctions.
Abstract: The authors investigated the presence of serotonin receptor type 1A (5-HT1A) as labeled by the specific ligand 3H-8 hydroxy-2-(di-N-propylamino)tetralin (3H-8-OH-DPAT) in saturation experiments, and the expression of the mRNA encoding them, in human peripheral blood mononuclear cells (PBMC). In situ hybridization experiments were performed as well. The results, showing that the binding of [3H]-8-OH-DPAT to lymphocyte membranes increased linearly up to 100 nM without reaching saturation, may indicate that the 3H-8-OH-DPAT was not specifically labeling the 5-HT1A receptor. By contrast, the expression studies revealed 5-HT1A mRNA in PBMC. These findings suggest that, despite the presence of mRNA, 5-HT1A receptors are not expressed in PBMC, at least in healthy controls.
