B-plexins control microtubule dynamics and dendrite morphology of hippocampal neurons

Semaphorins and their receptors plexins are implicated in various processes in the nervous system, but how B-plexins regulate the growth of dendrites remains poorly characterized. We had previously observed that Plexin-B1 and B3 interact with microtubule end-binding proteins (EBs) that are central adapters at growing microtubule tips, and this interaction is involved in neurite growth. Therefore, we hypothesized that plexins regulate microtubule dynamics and through that also dendritogenesis. The role of all three B-plexins was systematically examined in these processes. B-plexins and their ligand Semaphorin-4D influence the dynamics of microtubule tips both EB-dependently and independendently. EB3 as well as Plexin-B1, B2 and B3 turned out to have a significant role in the development of dendritic arbor of rat hippocampal neurons. Our results clearly indicate that semaphorin-plexin-EB pathway is one molecular mechanism how extracellular guidance cues are translated into intracellular mechanics. Taken together, Semaphorin-4D and B-plexins modulate the dynamic behavior of microtubule tips, and are therefore important in neurite growth.     

Plexin-B1 activates NF-κB and IL-8 to promote a pro-angiogenic response in endothelial cells

Background

The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium.

Methods/Principal Findings

Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma.

Conclusion/Significance

These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.     

Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration. We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras-mediated phosphatydylinositol 3-kinase activation, and beta(1) integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras-specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing beta(1) integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated beta(1) integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1-mediated R-Ras GAP activity controls cell migration by modulating the activity of beta(1) integrins.     

Protein tyrosine phosphatase SHP2 is involved in Semaphorin 4D-induced axon repulsion

Semaphorin-4D (Sema4D), a member of class 4 membrane-bound Semaphorins, acts as a chemorepellant to the axons of retinal ganglion cells and hippocampal neurons. Plexin-B1, a neuronal Sema4D receptor, associates with either one of receptor tyrosine kinases, c-Met or ErbB2, to mediate Sema4D-signaling. In contrast to this significance, the involvement of protein tyrosine phosphatases in Semaphorin-signaling remains unknown. We here show that Src homology 2-containing protein-tyrosine phosphatase 2 (SHP2) participates in Sema4D-signaling. SHP2 was localized in the growth cones of chick embryonic retinal ganglion neurons. Phenylarsine oxide, a protein tyrosine phosphatase inhibitor, suppressed Sema4D-induced contractile response in COS-7 cells expressing Plexin-B1. Ectopic expression of a phosphatase-inactive mutant of SHP2 in the retinal ganglion cells attenuated Sema4D-induced growth cone collapse response. A SHP1/2 specific inhibitor, 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), also suppressed this collapse response. These results suggest that SHP2-mediated tyrosine dephosphorylation is an important step in Sema4D-induced axon repulsion.     

Semaphorin 4D/Plexin-B1 Stimulates PTEN Activity through R-Ras GTPase-activating Protein Activity,Inducing Growth Cone Collapse in Hippocampal Neurons

Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 α activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.     

Sema4D/Plexin-B1 promotes the progression of osteosarcoma cells by activating Pyk2-PI3K-AKT pathway

Objectives:Osteosarcoma (OS) is one of the two most common malignant bone tumors among children and teens but it is still a rare disorder. Semaphorin 4D (Sema4D) has been reported to play a specific role in human cancers. The aim of this study was to explore the function of Sema4D in the tumorigenesis and development of OS.Methods:10 pairs of OS tissues and paracancerous normal tissues from human OS samples and OS cell lines were used. Western blot assay was performed to detect the protein expression of Sema4D, Plexin-B1, and associated proteins of Pyk2-PI3K/AKT pathway. To explore the effect of Sema4D in the progression of OS, we reduced the expression of Sema4D. The effect of Sema4D knockdown on cell proliferation was explored by CCK-8 assay and clone formation assay. The effect of Sema4D knockdown on cell migration and invasion was assessed by Transwell assay.Results:Sema4D was overexpressed in OS tissues and cell lines. Sema4D knockdown notably suppressed cell proliferation in OS cells. Cell migration and invasion were reduced by Sema4D knockdown. Sema4D/Plexin-B1 facilitated OS, progression by promoting Pyk2-PI3K/AKT pathway.Conclusion:Sema4D/Plexin-B1 promoted the development of OS so Sema4D might be a potential target of treatment for patients with OS.     

Semaphorin 4D/plexin-B1 induces endothelial cell migration through the activation of PYK2, Src, and the phosphatidylinositol 3-kinase-Akt pathway

Semaphorins are cell surface and secreted proteins that provide axonal guidance in neuronal tissues and regulate cell motility in many cell types. They act by binding a family of transmembrane receptors known as plexins, which belong to the c-Met family of scatter factor receptors but lack an intrinsic tyrosine kinase domain. Interestingly, we have recently shown that Plexin-B1 is highly expressed in endothelial cells and that its activation by Semaphorin 4D elicits a potent proangiogenic response (J. R. Basile, A. Barac, T. Zhu, K. L. Guan, and J. S. Gutkind, Cancer Res. 64:5212-5224, 2004). In searches for the underlying molecular mechanism, we observed that Semaphorin 4D-stimulated endothelial cell migration requires the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Surprisingly, we found that Plexin-B1 stimulates PI3K-Akt through the activation of an intracellular tyrosine kinase cascade that involves the sequential activation of PYK2 and Src. This results in the tyrosine phosphorylation of Plexin-B1, the rapid recruitment of a multimeric signaling complex that includes PYK2, Src, and PI3K to Plexin-B1 and the activation of Akt. These findings suggest that Plexin-B1 may achieve its numerous physiological functions through the direct activation of intracellular tyrosine kinase cascades.     

Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer

Bone density is controlled by interactions between osteoclasts, which resorb bone, and osteoblasts, which deposit it. The semaphorins and their receptors, the plexins, originally shown to function in the immune system and to provide chemotactic cues for axon guidance, are now known to play a role in this process as well. Emerging data have identified Semaphorin 4D (Sema4D) as a product of osteoclasts acting through its receptor Plexin-B1 on osteoblasts to inhibit their function, tipping the balance of bone homeostasis in favor of resorption. Breast cancers and other epithelial malignancies overexpress Sema4D, so we theorized that tumor cells could be exploiting this pathway to establish lytic skeletal metastases. Here, we use measurements of osteoblast and osteoclast differentiation and function in vitro and a mouse model of skeletal metastasis to demonstrate that both soluble Sema4D and protein produced by the breast cancer cell line MDA-MB-231 inhibits differentiation of MC3T3 cells, an osteoblast cell line, and their ability to form mineralized tissues, while Sema4D-mediated induction of IL-8 and LIX/CXCL5, the murine homologue of IL-8, increases osteoclast numbers and activity. We also observe a decrease in the number of bone metastases in mice injected with MDA-MB-231 cells when Sema4D is silenced by RNA interference. These results are significant because treatments directed at suppression of skeletal metastases in bone-homing malignancies usually work by arresting bone remodeling, potentially leading to skeletal fragility, a significant problem in patient management. Targeting Sema4D in these cancers would not affect bone remodeling and therefore could elicit an improved therapeutic result without the debilitating side effects.     

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

Background  

A wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.     

Estrogen-Dependent Proteolytic Cleavage of Semaphorin 4D and Plexin-B1 Enhances Semaphorin 4D-Induced Apoptosis during Postnatal Vaginal Remodeling in Pubescent Mice

Around the fifth week after birth, the vaginal cavity in female mouse pups opens to the overlaying skin. This postnatal tissue remodeling of the genital tract occurs during puberty, and it largely depends upon hormonally induced apoptosis that mainly occurs in the epithelium at the lower part of the mouse vaginal cavity. Previously, we showed that most BALB/c mice lacking the class IV Semaphorin (Sema4D) develop imperforate vagina and hydrometrocolpos; therefore, we reasoned that the absence of Sema4D-induced apoptosis in vaginal epithelial cells may cause the imperforate vagina. Sema4D signals via the Plexin-B1 receptor; nevertheless detailed mechanisms mediating this hormonally triggered apoptosis are not fully documented. To investigate the estrogen-dependent control of Sema4D signaling during the apoptosis responsible for mouse vaginal opening, we examined structural and functional modulation of Sema4D, Plexin-B1, and signaling molecules by analyzing both wild-type and Sema4D−/− mice with or without ovariectomy. Both the release of soluble Sema4D and the conversion of Plexin-B1 by proteolytic processing in vaginal tissue peaked 5 weeks after birth of wild-type BALB/c mice at the time of vaginal opening. Estrogen supplementation of ovariectomized wild-type mice revealed that both the release of soluble Sema4D and the conversion of Plexin-B1 into an active form were estrogen-dependent and concordant with apoptosis. Estrogen supplementation of ovariectomized Sema4D−/− mice did not induce massive vaginal apoptosis in 5-week-old mice; therefore, Sema4D may be an essential apoptosis-inducing ligand that acts downstream of estrogen action in vaginal epithelium during this postnatal tissue remodeling. Analysis of ovariectomized mice also indicated that Sema4D contributed to estrogen-dependent dephosphorylation of Akt and ERK at the time of vaginal opening. Based on our results, we propose that apoptosis in vaginal epithelium during postnatal vaginal opening is induced by enhanced Sema4D signaling that is caused by estrogen-dependent structural changes of Sema4D and Plexin-B1.     

MT1-MMP controls tumor-induced angiogenesis through the release of semaphorin 4D

The semaphorins are a family of proteins originally identified as regulators of axon growth that recently have been implicated in blood vessel development. The plexins are high affinity receptors for the semaphorins and are responsible for initiation of signaling upon ligation. Emerging evidence indicates that many human cancers overexpress Semaphorin 4D, which promotes neovascularization upon stimulating its receptor, Plexin-B1, on endothelial cells. However, to exert its pro-angiogenic functions, Semaphorin 4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. Here we show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but non-tumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo. These results suggest that the proteolytic cleavage of Semaphorin 4D may provide a novel molecular mechanism by which membrane type 1-matrix metalloproteinase controls tumor-induced angiogenesis.     

Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells

Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Here we show that Rnd1, a member of Rho family GTPases, directly interacted with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induced cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 did not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells was suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. We also found that Rnd1 promoted the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiated the Plexin-B1-mediated RhoA activation. We propose that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration.     

Plexin-B1, glycodelin and MMP7 expression in the human fallopian tube and in the endometrium

Background  

To study the expression of Plexin-B1, Glycodelin, and MMP7 during the menstrual cycle in the endometrium and in the fallopian tube.     

Immunomodulation by maternal autoantibodies of the fetal serotoninergic 5-HT<Subscript>4</Subscript>receptor and its consequences in early BALB/c mouse embryonic development

Background  

The presence of functional 5-HT₄ receptors in human and its involvement in neonatal lupus erythematosus (NLE) have prompted us to study the receptor expression and role during embryogenesis. Earlier we managed to demonstrate that female BALB/c mice immunized against the second extracellular loop (SEL) of the 5-HT₄ receptor gave birth to pups with heart block. To explain this phenomenon we investigated the expression of 5-HT₄ receptors during mouse embryogenesis. At the same time we looked whether the consequence of 5-HT₄ receptor immunomodulation observed earlier is in relation to receptor expression.     

Dopamine D4 receptor oligomerization--contribution to receptor biogenesis

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.2)R, D(4.4)R and D(4.7)R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D(4)R biogenesis and quality control in the endoplasmic reticulum plays an important role for D(4)R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D(4.4)R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D(4)R. The different polymorphic repeat variants of the D(4)R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D(4)Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.     

Memory retrieval in addiction: a role for miR-105-mediated regulation of D1 receptors in mPFC neurons projecting to the basolateral amygdala

Background

Drug addiction is a chronic brain disorder characterized by the compulsive use of drugs. The study of chronic morphine-induced adaptation in the brain and its functional significance is of importance to understand the mechanism of morphine addiction. Previous studies have found a number of chronic morphine-induced adaptive changes at molecular levels in the brain. A study from our lab showed that chronic morphine-induced increases in the expression of D1 receptors at presynaptic terminals coming from other structures to the basolateral amygdala (BLA) played an important role in environmental cue-induced retrieval of morphine withdrawal memory. However, the neurocircuitry where the increased D1 receptors are located and how chronic morphine increases D1 receptor expression in specific neurocircuits remain to be elucidated.

Results

Our results show that chronic morphine induces a persistent increase in D1 receptor expression in glutamatergic terminals of projection neurons from the medial prefrontal cortex (mPFC) to the BLA, but has no influence on D1 receptor expression in projection neurons from the hippocampus or the thalamus to the BLA. This adaptation to chronic morphine is mediated by reduced expression of miR-105 in the mPFC, which results in enhanced D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA. Ex vivo optogenetic experiments show that a chronic morphine-induced increase in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA results in sensitization of the effect of D1 receptor agonist on presynaptic glutamate release. mPFC to BLA projection neurons are activated by withdrawal-associated environmental cues in morphine-withdrawal rats, and overexpression of miR-105 in the mPFC leads to reduced D1 receptor induction in response to chronic morphine in glutamatergic terminals of the projection neurons from the mPFC to the BLA, and a reduction in place aversion conditioned by morphine withdrawal.

Conclusions

These results suggest that chronic morphine use induces a persistent increase in D1 receptors in glutamatergic terminals of projection neurons from the mPFC to the BLA via downregulation of miR-105 in the mPFC, and that these adaptive changes contribute to environmental cue-induced retrieval of morphine withdrawal memory.

     

Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy

Background  

Transient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established.     

Dopamine D4 receptor activation increases hippocampal gamma oscillations by enhancing synchronization of fast-spiking interneurons

Background

Gamma oscillations are electric activity patterns of the mammalian brain hypothesized to serve attention, sensory perception, working memory and memory encoding. They are disrupted or altered in schizophrenic patients with associated cognitive deficits, which persist in spite of treatment with antipsychotics. Because cognitive symptoms are a core feature of schizophrenia it is relevant to explore signaling pathways that potentially regulate gamma oscillations. Dopamine has been reported to decrease gamma oscillation power via D1-like receptors. Based on the expression pattern of D4 receptors (D4R) in hippocampus, and pharmacological effects of D4R ligands in animals, we hypothesize that they are in a position to regulate gamma oscillations as well.

Methodology/Principal Findings

To address this hypothesis we use rat hippocampal slices and kainate-induced gamma oscillations. Local field potential recordings as well as intracellular recordings of pyramidal cells, fast-spiking and non-fast-spiking interneurons were carried out. We show that D4R activation with the selective ligand PD168077 increases gamma oscillation power, which can be blocked by the D4R-specific antagonist L745,870 as well as by the antipsychotic drug Clozapine. Pyramidal cells did not exhibit changes in excitatory or inhibitory synaptic current amplitudes, but inhibitory currents became more coherent with the oscillations after application of PD168077. Fast-spiking, but not non-fast spiking, interneurons, increase their action potential phase-coupling and coherence with regard to ongoing gamma oscillations in response to D4R activation. Among several possible mechanisms we found that the NMDA receptor antagonist AP5 also blocks the D4R mediated increase in gamma oscillation power.

Conclusions/Significance

We conclude that D4R activation affects fast-spiking interneuron synchronization and thereby increases gamma power by an NMDA receptor-dependent mechanism. This suggests that converging deficits on fast-spiking interneurons may lead to decreased network function and thus aberrant gamma oscillations and cognitive decline in schizophrenia.     

Autoradiographic Localization of the Putative D4 Dopamine Receptor in Rat Brain

The putative dopamine D4 receptor protein in rat brain was labelled and quantified autoradiographically using two selective benzamides: [³H]YM-09151-2 which labels D2, D3 and D4 dopamine receptors and [³H]Raclopride which labels D2 and D3. The difference in densities of both ligands at saturable concentrations, show a regional distribution for the putative D4 receptor in the following rank order: hippocampus > caudate putamen > olfactory tubercle = substancia nigra > nucleus accumbens core > cerebral cortex > cerebellum. A calculated value of 0.34 pmol/mg protein was attributable to D4 receptor maximum capacity in caudate putamen and was obtained after subtracting the Bmax of the ligands. Our results show that the distribution of D4 receptor only partially overlaps with the D4 mRNA localization reported earlier and is not only associated to limbic structures but to motor areas as well.