A mental retardation gene,motopsin /neurotrypsin /prss12, modulates hippocampal function and social interaction

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions, including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long‐term memory formation in Drosophila. To understand motopsin’s function in the mammalian brain, motopsin knockout (KO) mice were generated. Motopsin KO mice did not have significant deficits in memory formation, as tested using the Morris water maze, passive avoidance and Y‐maze tests. A social recognition test showed that the motopsin KO mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin KO mice spent a longer time investigating a familiar mouse than wild‐type (WT) mice did. In a resident–intruder test, motopsin KO mice showed prolonged social interaction as compared with WT mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin KO mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP‐responsive element‐binding protein (CREB) in hippocampal neurons of WT mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons.     

Non-phosphorylated neurofilament protein immunoreactivity in adult and developing rat hippocampus: specificity and application in grafting studies

Neurofilament proteins are critical to the development and maintenance of neuronal shape in the nervous system. These proteins are developmentally regulated and several transition forms are expressed, prior to full neuronal stabilization. We have studied the spatial distribution and time course of expression of non-phosphorylated neurofilament protein (NPNFP) immunoreactivity in several preparations of rat hippocampus, using a mixture (SMI 311) of several monoclonal antibodies directed against NPNFP epitopes. Differential staining was observed in young and adult hippocampus. Large pyramidal neurons in CA3 and CA4 subfields were strongly immunoreactive in adult hippocampus whereas the smaller CA1 pyramidal neurons, most interneurons and dentate granule cells were immunonegative. SMI 311 staining initially appeared at postnatal day (P) 5 with positive staining in apical dendrites and soma in a few pyramidal neurons in CA3, but almost reached the adult pattern by P10. Compared to adult hippocampus, the number of immunoreactive interneurons in all subfields appeared increased at P10 and P15. In cultures of embryonic hippocampus, all neurons, regardless of their morphology, were SMI 311 positive, suggesting loss of differential expression in tissue culture conditions. However, SMI 311 expression in fetal hippocampal neurons grafted to adult hippocampus was similar to hippocampal neurons which had developed in situ. These results suggest that SMI 311 antibody identifies a distinct group of primarily CA3 and CA4 pyramidal cells in adult hippocampus. The application of SMI 311 immunostaining appears suitable for identification of large CA3 and CA4 pyramidal neurons within hippocampal transplants grafted to adult CNS but not in tissue culture.     

Spine distribution along the apical dendrites of the pyramidal neurons in Down's syndrome

Summary The number of spines along the apical dendrites of the pyramidal neurons in the hippocampal and cingulate gyri were counted on Golgi preparations of the brains of seven cases of Down's syndrome; they did not show neuropathological senile changes. The number of spines in the middle and distal segments of the apical dendrites of Down's syndrome group were significantly fewer than in those of the control group. The number of spines in aged noncharacteristics mentally retarded cases were not decreased compared with the controls.The diminution of spines in Down's syndrome is considered to be rather specific in this condition and is not a common finding in other types of mental retardation. It might also be one of the early changes of dendritic degeneration related to senile plaque formation.This study was supported in part by a scholarship from the Max-Plank Society on the MPI for Psychiatry, Munich     

Variations in the activity and sexual dimorphism of hippocampal pyramidal neurons during the postnatal development of the rat

The CA 1 pyramidal neurons of the dorsal and ventral hippocampus as also the neurons of the granular layer of the parietal cortex, which were investigated in comparison, show karyovolumetrically revealed significant variations of their activities during the postnatal development of the rat (between the 5th and 60th postnatal day). These variations represent the total expression of the gene activities related to the development and differentiation as also to the synchronously requested specific performances of the neurons for the organism. They are characterized by a more or less distinct regional specificity as also a sexual dimorphism--especially distinct during the first 3...4 weeks of postnatal development--beside several distinct principal coincidences. Characteristic are the peaks of activity at the 10th and 20th day of postnatal life and additionally at the 45th day in male animals. The activation of the hippocampal pyramidal neurons as also of the neurons in the granular layer of the parietal cortex from day 5 to day 10 of the postnatal development, which comes to appearance as an increase of cell nuclear volume between 35,2 and 142,5 per cent, coincides with the growing period of the central nervous system, which lasts in the rat till the 10th postnatal day, and with the postnatal initial phase of several extracerebral developmental, differentiation and maturation processes. The variations of neuronal activities occurring after the 10th day and ceasing in the 4th week of the postnatal development coincide with the maturation period of the central nervous system and of the extracerebral systems. The peak of activity appearing around the 45th day of life in male rats seems to be related to the puberty. The sexual dimorphism of the hippocampal pyramidal neurons as also of the neurons in the granular layer of the parietal cortex, realized in the cell nuclear volume resp. neuronal activity, is apparently the reflection of the sexual specific differentiation of the central nervous system and is a partial phenomenon of the complex dimorphism of the both sexes. It shows also a regional specific character.     

Urokinase-type Plasminogen Activator Expression by Neurons and Oligodendrocytes During Process Outgrowth in Developing Rat Brain

The expression of tissue- and urokinase-type plasminogen activators has been studied in developing cerebellum, hippocampus, cerebral cortex, olfactory bulb and olfactory mucosa of the rat by in situ hybridization. All identifiable neurons express urokinase mRNA from an early stage in their development, and this expression appears to coincide with the onset of axogenesis. For cerebellar granule cells, both axonal growth and urokinase expression are initiated before they migrate from the external granule layer; for the majority of neocortical neurons, however, both processes are commenced after the cells have migrated to the cortical plate. Neurons continue to express this protease in the adult. The large projection neurons exhibit the highest levels of message, the smaller interneurons having much lower levels except for hippocampal granule cells, which have notably high levels of expression. Glial cells generally do not express urokinase message, except for transient expression by oligodendrocytes in developing fibre tracts during the period of myelination. Thus for both neurons and oligodendrocytes, the onset of urokinase-type plasminogen activator expression coincides with their initiation of major process outgrowth, although neurons maintain this expression in the adult, possibly to retain a degree of synaptic plasticity. In contrast, although high levels of message for the related protease, tissue plasminogen activator, are found in the embryonic floor plate, in postnatal brain it is abundantly expressed only by ventricular ependymal cells and by cells in connective tissue surrounding the olfactory nerve.     

Neurohistologic studies of cortical pyramidal neurons in the rat following rehabilitation of early postnatal malnutrition

The experimental animals were 60 days old rats which had been suffering from undernutrition during day 1 till day 20 of their early postnatal life. The recovery from the retardation in brain development induced by the experiment was investigated by quantitative light microscopical methods in GOLGI impregnated material. In pyramidal neurons of lamina III or V of the anterior cingulate cortex several neuronal parameters were estimated in a single dendritic field (EDF) indicative for the development of the dendritic tree and spine distribution of these neurons. The results demonstrate a sufficient recovery in those cases when the rehabilitation is beginning already within the period of intensive brain growth. The duration of this growth spurt is prolongated in the experimental animals. Therefore, after the starvation period the brain is supposed to have some potency for further neuronal differentiation, also beyond the 20th day of life. This fact is considered to be decisive for a full recovery. After the starvation period at day 20 the lamina V pyramidal neurons were found to have developed a significantly smaller dendritic tree (minus 15%) equipped with a strongly reduced spine number (minus 30%). After the recovery time of 40 days the control values were reached, moreover, the spine density in apical dendrites was enhanced in comparison to controls. Likewise, in the lamina III pyramidal neurons of experimental animals, the apical dendritic tree was shortened by 25-30%, the basal dendritic tree by 10% at the end of the undernutrition period. However, after recovery of 40 days the dendritic tree of these neurons developed a significant overshoot of 15-20% in comparison to controls. The spine number at basal dendrites was similar to controls, but at apical dendrites significantly higher. The differences in the recovery pattern of both neuron types are explained by the hypothesis that the lamina III pyramidal neurons are phylogenetically younger and, therefore, have a considerably longer postnatal differentiation phase. Thus, they show a stronger retardation in growth by the undernutrition. However, during rehabilitation and recovery they show a larger increment in growth and differentiation.     

Constitutive Ras activity induces hippocampal hypertrophy and remodeling of pyramidal neurons in synRas mice

The small G protein Ras, which is involved critically in neurotrophic signal transduction, has been implicated in neuronal plasticity of both the developing and the adult nervous systems. In the present study, the cumulative effects of constitutive Ras activity from early in postnatal development into the adult upon the morphology of hippocampal pyramidal neurons were investigated in synRas mice overexpressing Val12-Ha-Ras postmitotically under the control of the rat synapsin I promoter. In synRas mice, stereologic investigations revealed hypertrophy of the hippocampus associated with an increase in perikaryal size of pyramidal neurons within the CA2/CA3 region and the gyrus dentatus. Morphometric analyses of Lucifer Yellow-filled CA1 pyramidal neurons, in addition, demonstrated considerable expansion of dendritic arbors. The increase in basal dendritic size was caused primarily by alterations of intermediate and distal segments and was associated with an enlarged dendritic surface. Apical dendrites showed similar but more moderate changes, which were attributed mainly to elongation of terminal segments. Sholl analyses illustrated higher complexity of both basal and apical trees. Despite significant morphologic alterations, dendritic arbors preserve their major design principles. The synaptic density within the stratum radiatum of CA1 remained unchanged; however, increases in the total hippocampal volume and in apical dendritic size imply an increment in the absolute number of synaptic contacts. The data presented here suggest a critical involvement of Ras dependent signaling in morphoregulatory processes during the maturation and in the maintenance of hippocampal pyramidal neurons.     

Serine protease inhibitors: novel therapeutic targets for stroke?

Although the thrombolytic activity of tissue-type plasminogen activator (t-PA) may be beneficial in the acute treatment of stroke, recent studies have suggested that this serine protease could also play a critical role in determining the extent of neuronal death after injury to the central nervous system (CNS). This hypothesis is based on several experimental results: t-PA-deficient mice are resistant to excitotoxic neuronal death induced by the intrahippocampal injection of kainate; the infarct volume induced by occlusion of the middle cerebral artery is reduced in t-PA knockout mice; and the intravenous injection of t-PA can under certain circumstances potentiate the infarct volume in animals subjected to middle cerebral artery occlusion. In the CNS, the serine proteases have been identified to occur both in neurons and glial cells. Their enzymatic activity regulates the balance between the accumulation and the degradation of the extracellular matrix. They are involved in many physiologic functions, ranging from synaptic outgrowth during perinatal development to plasticity in adults. For instance, thrombin and t-PA are known to modulate neurite outgrowth and tissue remodeling in the early stages of development. In the adult brain, t-PA may contribute to the late phase of long-term potentiation and to the subsequent synaptic growth in the hippocampal mossy fiber pathway. This balance between the degradation and accumulation of the extracellular matrix may also be integral to various pathologic processes involved in acute brain injury. For example, compounds that modulate the activity of serine proteases exhibit neuroprotective activity. Based on the above, numerous studies have focused on the production and modulation of the endogenously produced serine protease inhibitors, termed serpins, such as type 1 plasminogen activator inhibitor, neuroserpin, and protease nexin-1. In the present review, we will discuss the need to distinguish between the potentially neurotoxic effects of t-PA and its beneficial effect on reperfusion. We will present data supporting the idea that the modulation of serine protease activity may represent a novel and efficient strategy for the treatment of acute cerebral injury in humans.     

Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice

Semaphorin‐3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin‐1 and plexin‐A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as “proximal bifurcation.” In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal‐like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin‐dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development. J. Comp. Neurol. 516:360–375, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons.     

Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons

Mice lacking serotonin receptor 1A (Htr1a) display increased anxiety behavior that depends on the expression of the receptor in the forebrain during the third to fifth postnatal weeks. Within the forebrain, Htr1a is prominently expressed in the soma and dendrites of CA1 pyramidal neurons of the hippocampus and these cells undergo rapid dendritic growth and synapse formation during this period. Consistent with a possible role of Htr1a in synaptic maturation, CA1 pyramidal neurons in the knockout mice show increased ramification of oblique dendrites. These findings suggest that Htr1a may shape hippocampal circuits by directly modulating dendritic growth. Here we show that pharmacological blockade of the receptor during the third to fifth postnatal weeks is sufficient to reproduce the increased branching of oblique dendrites seen in knockout mice. Using dissociated hippocampal cultures we demonstrate that serotonin functions through Htr1a to attenuate the motility of dendritic growth cones, reduce their content of filamentous actin and alter their morphology. These findings suggest that serotonin modulates actin cytoskeletal dynamics in hippocampal neurons during a limited developmental period to restrict dendritic growth and achieve a long‐term adjustment of neural connectivity.     

Polarized distribution of alpha5 integrin in dendrites of hippocampal and cortical neurons

The distribution of immunoreactivity for the alpha5 subunit of the fibronectin receptor was evaluated in adult rat brain with particular interest in the cellular localization of immunostaining in the hippocampal formation and neocortex. Beyond localization to neuronal perikarya and short dendritic fragments within most brain areas, alpha5 immunoreactivity (-ir) was particularly dense within primary apical dendrites of pyramidal cells in both hippocampus and neocortex and within the dendritic arbors of cerebellar Purkinje cells. In hippocampal and cortical pyramidal cells, immunostaining was clearly polarized: alpha5-ir was not detectable in basal dendrites in hippocampal neurons and was limited to proximal arbors or absent from basal dendrites in pyramidal cells in superficial and deep layers of neocortex. Beyond this, alpha5-ir was distributed within the dendritic ramifications of the dentate gyrus granule cells and within perikarya and dendrites of occasional nonpyramidal neurons. Developmental studies demonstrated that, in both hippocampus and neocortex, alpha5-ir appears first within perikarya and is distributed to dendrites during the second postnatal week. These results are in accord with the broad hypothesis that integrins contribute to apical-basal differences in dendrites and that the integrin fibronectin (alpha5beta1) receptor, in particular, contributes to some late developing features of dendritic structure or function.     

Differential Expression of Synaptophysin and Synaptoporin During Pre- and Postnatal Development of the Rat Hippocampal Network

The closely related synaptic vesicle membrane proteins synaptophysin and synaptoporin are abundant in the hippocampal formation of the adult rat. But the prenatal hippocampal formation contains only synaptophysin, which is first detected at embryonic day 17 (E17) in perikarya and axons of the pyramidal neurons. At E21 synaptophysin immunoreactivity extends into the apical dendrites of these cells and in newly formed terminals contacting these dendrites. The transient presence of synaptophysin in axons and dendrites suggests a functional involvement of synaptophysin in fibre outgrowth of developing pyramidal neurons. Synaptoporin expression parallels the formation of dentate granule cell synaptic contacts with pyramidal neurons: the amount of hippocampal synaptoporin, determined in immunoblots and by synaptoporin immunostaining of developing mossy fibre terminals, increases during the first postnatal week. Moreover, in the adult, synaptoporin is found exclusively in the mossy fibre terminals present in the hilar region of the dentate gyrus and the regio inferior of the cornu ammonis. In contrast, synaptophysin is present in all synaptic fields of the hippocampal formation, including the mossy fibre terminals, where it colocalizes with synaptoporin in the same boutons. Our data indicate that granule neuron terminals differ from all other terminals of the hippocampal formation by the presence of both synaptoporin and synaptophysin. This difference, observed in the earliest synaptic contacts in the postnatal hippocampus and persisting into adult life, suggests distinct functions of synaptoporin in these nerve terminals.     

Translation of an integral membrane protein in distal dendrites of hippocampal neurons

Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons.     

Dock4 regulates dendritic development in hippocampal neurons

Dendrite development is required for establishing proper neuronal connectivity. Rho-family small GTPases have been reported to play important roles in the regulation of dendritic growth and morphology. However, the molecular mechanisms that control the activities of Rho GTPases in developing dendrites are not well understood. In the present study we found Dock4, an activator of the small GTPase Rac, to have a role in regulating dendritic growth and branching in rat hippocampal neurons. Dock4 is highly expressed in the developing rat brain, predominantly in hippocampal neurons. In dissociated cultured hippocampal neurons, the expression of Dock4 protein is up-regulated after between 3 and 8 days in culture, when dendrites begin to grow. Knockdown of endogenous Dock4 results in reduced dendritic growth and branching. Conversely, overexpression of Dock4 with its binding partner ELMO2 enhances the numbers of dendrites and dendritic branches. These morphological effects elicited by Dock4 and ELMO2 require Rac activation and the C-terminal Crk-binding region of Dock4. Indeed, Dock4 forms a complex with ELMO2 and CrkII in hippocampal neurons. These findings demonstrate a new function of the Rac activator Dock4 in dendritic morphogenesis in hippocampal neurons.     

Ultrastructural characterization and GAD co-localization of somatostatin-like immunoreactive neurons in CA1 of rabbit hippocampus

Immunocytochemical techniques have been used to identify a striking interneuronal population which is immunoreactive for the peptide, somatostatin. The cell population, which is seen most densely in stratum oriens and at the oriens/alveus border of the CA1 region of rabbit hippocampus, was characterized in light and electron microscopic observations. The cells have dendrites which extend parallel to and into the alveus, with occasional processes ascending through stratum pyramidale toward the hippocampal fissure. The dendrites receive numerous synaptic contacts directly onto aspinous dendritic shafts. Axon collaterals ramify profusely within the pyramidale region, and among the proximal apical and basal pyramidal cell dendrites in areas of stratum radiatum and stratum oriens. Somatostatin-like immunoreactive terminals make synaptic contact, primarily of the symmetric type, with the somata and proximal dendrites of pyramidal neurons. Somatostatin-like neurons are found at approximately equal density in the hippocampus of immature (8 days postnatal) and mature (30 days postnatal) rabbit. Double-labelling techniques, to identify both somatostatin-like and glutamic acid decarboxylase (GAD) immunoreactive neurons, demonstrated that a large proportion of the somatostatin neurons were also GABAergic.