Synovial Sarcoma Microvesicles Harbor the SYT-SSX Fusion Gene Transcript: Comparison of Different Methods of Detection and Implications in Biomarker Research.

Background. Synovial sarcoma is an aggressive soft-tissue malignancy. This study examines the presence of the SYT-SSX fusion transcript in synovial sarcoma microvesicles as well as its potential role as a biomarker for synovial sarcoma. Patients and Methods. Microvesicle release of synovial sarcoma cells was examined by transmission electron microscopy. RNA-content was analyzed by qPCR, nested PCR, nested qPCR, and droplet digital PCR to compare their sensitivity for detection of the SYT-SSX fusion gene transcript. Whole blood RNA, RNA of mononuclear cells, and microvesicle RNA of synovial sarcoma patients were analyzed for the presence of the fusion gene transcripts. Results. Electron microscopic analysis revealed synovial sarcoma cells releasing membrane-enclosed microvesicles. In vitro, the SYT-SSX fusion gene transcript was detected in both synovial sarcoma cells and microvesicles. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA. In contrast, the fusion gene transcript was not detected in peripheral blood cells and microvesicles of synovial sarcoma patients. Conclusion. Synovial sarcoma cells release microvesicles harboring the SYT-SSX fusion transcript. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA; however, more sensitive assays are needed to detect cancer-specific microvesicles in the peripheral blood of cancer patients.

P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro.

Extracellular ATP might act as a trophic factor on growing axons during development of the CNS via P2 receptors. In the present study the postnatal presence of selected P2 receptor subtypes was analyzed and their putative trophic capacity in entorhino-hippocampal slice co-cultures of mouse brain was tested. The effect of the P2 receptor ligands 2-methylthioadenosine-5'-triphosphate (P2X/Y receptor agonist) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2X/Y receptor antagonist) on axonal growth and fiber density of biocytin-labeled hippocampal projections was compared both with untreated cultures and with cultures treated with artificial cerebrospinal fluid. After 10 days in vitro, double immunofluorescence labeling revealed the expression of P2X(1), P2X(2), P2X(4) as well as P2Y(1) and P2Y(2) receptors in the examined regions of entorhinal fiber termination. Further, quantitative analysis of identified biocytin-traced entorhinal fibers showed a significant increase in fiber density in the dentate gyrus after incubation of the slices with the P2 receptor agonist 2-methylthioadenosine-5'-triphosphate. This neurite outgrowth promoting effect was completely abolished by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Our in vitro data indicate that ATP via its P2X and P2Y receptors can shape hippocampal connectivity during development.

Myelination in the hippocampus during development and following lesion.

Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate for outgrowing axons. Therefore, one major factor which accounts for the lack of axonal regeneration in the mature brain is myelin. Here we report on the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal lesion with the myelin-specific marker Black Gold. Although entorhinal axons enter the hippocampal formation at embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibers in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long-lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganization pha-ses in the hippocampus. In contrast, electroconvulsive seizures resulted in brief demyelination and a faster recovery time course. In conclusion, we could show that the appearance of mature axons in the hippocampus is temporally regulated during development. In the adult hippocampus, demyelination was found after anterograde degeneration and also following seizures, suggesting that independent types of insult lead to demyelination. Reappearing mature axons were found in the hippocampus following axonal sprouting. Therefore, our quantitative analysis of mature axons and myelination effectively reflects the readjusted axonal density and possible electrophysiological balance following lesion.

Modulation of electrically evoked acetylcholine release in cultured rat septal neurones.

The electrically evoked release of acetylcholine and its modulation via auto- and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D-lysine-coated glass coverslips. They developed a dense network of non-neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre-incubated with [3H]choline (0.1 micromol/L), superfused with modified Krebs-Henseleit buffer at 25 degrees C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90-100 mA). The electrically evoked overflow of [3H] from the cells consisted of approximately 80% of authentic [3H]Ach, was largely Ca2+-dependent and tetrodotoxin sensitive, and hence represents an action potential-evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonist added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1- and opioid mu-receptors, could be detected, whereas delta-opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2-week-old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and mu-opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones.

Postnatal development of opioid receptors modulating acetylcholine release in hippocampus and septum of the rat.

The postnatal development of presynaptic opioid receptors inhibiting the release of acetylcholine (ACh) was studied in rat brain hippocampus, medial septum (MS) and diagonal band of Broca (DB). To this end, the corresponding brain slices (350 microm thick) of rats of various postnatal ages (postnatal day 4 [P4] to P16, and adult) were preincubated with [(3)H]choline and stimulated twice for 2 min (S(1), S(2): at 3 Hz, 2 ms, 60 mA) during superfusion with physiological buffer containing hemicholinium-3. In parallel, the activity of choline acetyltransferase (ChAT) was determined in crude homogenates of the tissues as a marker for the development of cholinergic neurons. At any postnatal age, the electrically evoked overflow of tritium from slices preincubated with [(3)H]choline was highest in the DB, followed by the MS and the hippocampus. The evoked [(3)H]overflow increased with postnatal age, reached about 50% (MS, DB) or 30% (hippocampus) of the corresponding adult levels at P16 and correlated significantly with the corresponding ChAT activities. Presence of the preferential mu-opioid receptor agonist DAMGO during S(2) significantly inhibited the evoked overflow of tritium already at P4 in DB and MS, whereas in the hippocampus significant inhibitory effects were first observed at P8 only. Moreover, adult levels of inhibition due to DAMGO were reached at P16 in the DB and MS but not in the hippocampus. In septal areas, also the effect of the preferential delta-opioid receptor agonist DPDPE on the evoked [(3)H]overflow was studied: in contrast to DAMGO, however, significant inhibitory effects of DPDPE were first observed at P12 only. In conclusion, the postnatal development of presynaptic mu-opioid receptors on cholinergic neurons in the DB and MS starts earlier than in the hippocampus and precedes that of presynaptic delta-opioid receptors.

Role of afferent innervation and neuronal activity in dendritic development and spine maturation of fascia dentata granule cells.

By using slice cultures of hippocampus as a model, we have studied the development of dendritic spines in fascia dentata granule cells. We raised the question as to what extent spine development is dependent on a major afferent input to these neurons, the fibers from the entorhinal cortex and neuronal activity mediated by these axons. Our results can be summarized as follows: (i) the entorhino-hippocampal projection develops in an organotypic manner in co-cultures of entorhinal cortex and hippocampus. Like in vivo, entorhinal fibers, labeled by anterograde tracing with biocytin, terminate in the outer molecular layer of the fascia dentata. (ii) The layer-specific termination of entorhinal fibers is not altered by the blockade of neuronal activity with tetrodotoxin. Likewise, the differentiation of the dendritic arbor of postsynaptic granule cells does not require neuronal activity. Blockade of neuronal activity did not affect the mean spine number of granule cell dendrites in entorhino-hippocampal co-cultures, but led to a relative increase in thin, long filiform spines that are characteristic of immature neurons. (iii) The maturation of the granule cell dendritic arbor is, however, controlled by the afferent fibers from the entorhinal cortex in an activity-independent manner. In single slice cultures of hippocampus lacking entorhinal input, Golgi-impregnated granule cells have much shorter, less branched dendrites when compared with granule cells in entorhino-hippocampal co-cultures. This reduction in dendritic length in granule cells lacking entorhinal input results in a lower mean total number of spines per neuron, but the mean number of spines per microm is not reduced in the absence of entorhinal innervation. These results indicate that innervation by fibers from the entorhinal cortex, but not neuronal activity mediated via these axons, is essential for the normal development of the granule cell dendritic arbor. Neuronal activity is required, however, for the maturation of dendritic spines.

Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus.

The transcription factors neuronal helix-loop-helix protein (NEX)/mammalian atonal homolog 2 (Math-2), BETA2/neuronal determination factor (NeuroD), and NeuroD-related factor (NDRF)/NeuroD2 comprise a family of Drosophila atonal-related basic helix-loop-helix (bHLH) proteins with highly overlapping expression in the developing forebrain. The ability of BETA2/NeuroD and NDRF to convert ectodermal cells into neurons after mRNA injection into Xenopus oocytes suggested a role in specifying neuronal cell fate. However, neuronal bHLH genes are largely transcribed in CNS neurons, which are fully committed. Here we analyze a defect in mice lacking BETA2/NeuroD, and in NEX*BETA2/NeuroD double mutants, demonstrating that bHLH proteins are required in vivo for terminal neuronal differentiation. Most strikingly, presumptive granule cells of the dentate gyrus are generated but fail to mature, lack normal sodium currents, and show little dendritic arborization. Long-term hippocampal slice cultures demonstrate secondary alterations of entorhinal and commissural/associational projections. The primary developmental arrest appears to be restricted to granule cells in which an autoregulatory system involving all three neuronal bHLH genes has failed.

Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies.

Cajal-Retzius (CR) cells are characteristic horizontally orientated, early-generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino-hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde-fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new-born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino-hippocampal slice cocultures using infrared videomicroscopy in combination with the patch-clamp technique. In double-labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino-hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus.

Blockade of neuronal activity alters spine maturation of dentate granule cells but not their dendritic arborization.

Organotypic co-cultures of the entorhinal cortex and hippocampus were examined to determine the role of the entorhinal fibers in the dendritic development and formation of spines of dentate granule cells. Quantitative analysis of Golgi-impregnated granule cells in single hippocampal cultures and co-cultures with the entorhinal cortex revealed that the presence of entorhinal fibers promoted the elongation and differentiation of the target granule cell dendrites. This was accompanied by an increase in the total number of spines. The contribution of neuronal activity to this afferent-mediated dendritic development was tested by chronic application of the sodium channel blocker tetrodotoxin for 20 days in vitro. Tracing with biocytin showed that the formation of the entorhinohippocampal pathway was unaffected by the blockade of neuronal activity. The dendritic arbor of cultured granule cells and the number of dendritic spines did not differ between tetrodotoxin-treated slices and untreated controls. However, there was a significant increase in the relative number of filiform spines on granule cell dendrites in tetrodotoxin-treated co-cultures. Such filiform spines are a characteristic feature of immature neurons. These results suggest the cooperation of two mechanisms in the dendritic development of dentate granule cells: the specific afferent-mediated dendritic arborization and the activity-dependent maturation of spines.

The hippocampus of the reeler mutant mouse: fiber segregation in area CA1 depends on the position of the postsynaptic target cells.

Area CA1 of the rodent hippocampus is characterized by a highly lamina-specific and nonoverlapping termination of afferent fiber tracts. Entorhinal fibers terminate in stratum lacunosum-moleculare and commissural/associational fibers terminate in strata radiatum and oriens. It has been hypothesized that this fiber lamination depends on specific signals for the different afferent fiber tracts that are located on distinct dendritic segments of the postsynaptic neuron. In order to test this hypothesis, entorhinal and commissural/associational afferents to Ammon's horn were investigated in the adult reeler mutant mouse, in which developmental cell migration defects have disrupted the normal array of cells. Golgi staining revealed a deep and a superficial principal cell layer in the mutant. The morphology of the cells located in the deep pyramidal cell layer was considerably abnormal, whereas most cells located in the superficial pyramidal cell layer showed an almost normal cellular and dendritic morphology. Anterograde tracing with Phaseolus vulgaris leukoagglutinin revealed that the duplication and disorganization of the pyramidal cell layer in area CA1 are mirrored by the duplication and disruption of afferent fiber termination zones. In the zone above the abnormal deep pyramidal cell layer, i.e., between the two cell layers, entorhinal fibers as well as commissural/associational fibers terminate and intermingle. In contrast, in the zone above the fairly normal superficial pyramidal cell layer, entorhinal and commissural/associational fibers retain their normal fiber segregation. These data suggest that the normal laminar organization of the murine hippocampus depends on positional cues presented by their target cells.

Postnatal development of muscarinic autoreceptors in the rat brain: lateral and medial septal nuclei and the diagonal band of Broca.

The postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors was studied in the lateral septum (LS), the medial septum (MS) and the diagonal band of Broca (DB) of the rat brain. To this end, slices (350 micrometer thick) containing these brain regions from rats of various postnatal ages (postnatal day 3 [P3] to P16, and adult) were pre-incubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 3 Hz) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In addition, the activity of choline acetyltransferase (ChAT, in crude homogenates) was determined as a marker for the development of cholinergic functions. At any postnatal age, the electrically-evoked overflow of tritium from slices pre-incubated with [3H]choline was highest in the DB, followed by the MS whereas in slices containing the LS, it was only small. In all septal regions, the evoked [3H]overflow was Ca2+-dependent and tetrodotoxin-sensitive at P3. It increased with postnatal age and reached about 60% of the corresponding adult levels at P16. Presence of the muscarinic agonist oxotremorine (1 microM) during S2 significantly inhibited the evoked overflow of tritium beginning from P5: no significant effect was detected at P3. The ACh esterase inhibitor physostigmine (1 microM) exhibited significant inhibitory effects from P13 onwards, whereas the muscarinic antagonist atropine (1 microM) did not change the evoked ACh release. The activity of ChAT, as measured for these septal regions at various postnatal ages, correlated well with the [3H]overflow induced by electrical stimulation. In conclusion, (1) electrically-evoked release of ACh was measured for the first time in three septal subregions; (2) the postnatal development of the presynaptic cholinergic functions: ChAT activity, ACh release and muscarinic autoreceptors occurs almost synchronously in these regions of the septal complex and parallels that in the hippocampal formation; (3) as in the hippocampus, the postnatal development of autoreceptors was delayed with respect to the exocytotic release of ACh.

Glutamatergic control of the expression of the proenkephalin gene in rat frontoparietal cortical slice cultures.

The expression of the proenkephalin (PEnk) gene in rat neocortex develops during the first two postnatal weeks in an outside first-inside last mode that is opposite to the gradient of neurogenesis. To test whether the distribution of PEnk gene expression depends on the formation of the local circuitry, we examined the role of glutamate neurons in the expression of the gene in slice cultures of rat frontoparietal cortex. In situ and Northern blot hybridization were used for analysis. In slices explanted at postnatal day 6, the neuronal expression of the PEnk gene developed as in vivo. The expression responded to glutamate receptor agonists and antagonists in a time-dependent manner. After 2 days in vitro the expression of the gene was only enhanced by N-methyl-D-aspartate receptors, whereas after 7 days in vitro AMPA receptors also regulated the expression. We concluded that glutamate neurons are involved in the development and maintenance of the PEnk gene expression in the neocortex.

Axotomy-induced c-JUN expression in young medial septal neurons is regulated by nerve growth factor.

In the present study we investigated the axotomy-induced expression of the proto-oncogene c-jun in young rat medial septal neurons and its regulation by nerve growth factor. First, medial septal neurons were retrogradely labelled by Fast Blue injection into the hippocampus at postnatal day 1 (P1). Rats of different developmental ages (P6, P9, P14, P21, P28 and P42) were then subjected to bilateral fimbria-fornix transection resulting in the axotomy of septohippocampal projection neurons. After the lesion, c-JUN immunoreactivity was observed in the nuclei of axotomized medial septal neurons of all stages examined, suggesting that c-JUN induction is an age-independent feature of axotomized medial septal neurons. Double immunolabelling for choline acetyltransferase and c-JUN or parvalbumin and c-JUN, respectively, revealed that both cholinergic and GABAergic septohippocampal projection neurons express c-JUN after axotomy. In addition, a co-localization of immunostaining for c-JUN and the neuropeptide galanin was found after lesion, as both proteins were induced in the same medial septal neurons following fimbria-fornix transection. Next, the regulation of c-JUN expression in axotomized medial septal neurons was studied in organotypic cultures of the medial septum. Axotomized medial septal neurons in culture did not express c-JUN in contrast to the in vivo situation. With the concept that nerve growth factor suppresses c-JUN expression, slice cultures of the medial septum were treated with antibodies against nerve growth factor. This treatment caused a dose-dependent increase in c-JUN-positive cells in these slice cultures. Simultaneous addition of nerve growth factor and antibodies against nerve growth factor resulted in the reversal of this effect. These data suggest an age-independent induction of c-JUN in axotomized medial septal neurons and its regulation by nerve growth factor.

Postnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system. I. Axon terminal region: hippocampus.

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the rat hippocampus. To this end, hippocampal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the cholinergic ingrowth. In hippocampal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.11 (P3) to 0.81% of tissue 3H (P16), the latter value being still much lower than that of hippocampal slices from adult rats (2.89% of tissue 3H). Already at P3 the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release. The muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in hippocampal slices with increasing effectivity from P4 to P16; no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine and the muscarinic antagonist atropine (1 microM, each) exhibited significant inhibitory and facilitatory effects, respectively, only at P15-16. The specific activities of both hippocampal HACU (pmoles/mg protein/min) and ChAT (nmoles/mg protein/min) continuously increased from P3 to P16. It is concluded (1) that cholinergic nerve terminals arriving at the hippocampal formation during postnatal ingrowth are already endowed with the apparatus for action potential-induced, Ca(2+)-sensitive (exocytotic) ACh release; (2) that, in contrast, the expression of presynaptic muscarinic autoreceptors on these cholinergic axon terminals is delayed; and (3) that autoinhibition due to endogenous ACh develops even later, probably when the density of presynaptic terminals in the hippocampus and hence, the concentration of released ACh has reached a suprathreshold value.

Postnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system. II. Cell body region: septum.

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the cell body region of the septohippocampal cholinergic pathway. To this end, septal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the development of cholinergic functions. In septal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.31% (P3) to 2.10% of tissue 3H (P16), the latter value being still lower than that of septal slices from adult rats (3.46% of tissue 3H). Already at P3, the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release early after birth. Presence of the muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in septal slices beginning from P5: no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine (1 microM) exhibited significant inhibitory effects from P13 onwards. The muscarinic antagonist atropine (1 microM) enhanced the evoked ACh release only in septal tissue from adult rats. The specific activities of HACU, or ChAT showed a 2- or 8-fold increase, respectively, from P3 to P16. In conclusion, presynaptic cholinergic functions seem to develop almost in parallel both in the cell body and the target area of the septohippocampal projection: also in the septal region nerve terminals on axon collaterals are endowed very early (at least at P3) with the apparatus for action potential-induced, exocytotic release of ACh. In contrast, the appearance of feedback inhibition via presynaptic muscarinic autoreceptors is delayed. Autoinhibition due to endogenously released ACh can be detected only later, most probably when endogenous ACh concentrations in the septal nuclei have reached a threshold value.

Hippocampal mossy fiber sprouting is not impaired in brain-derived neurotrophic factor-deficient mice.

In human temporal lobe epilepsy, a loss of hilar neurons followed by the sprouting of recurrent mossy fiber collaterals and the reinnervation of free synaptic sites on granule cell dendrites are discussed as possible mechanisms underlying hippocampal hyperexcitability. Dentate granule cells have been shown to upregulate brain-derived neurotrophic factor (BDNF) as well as TrkB, the high-affinity receptor for BDNF, in response to limbic seizures. This raised the possibility that BDNF is an important factor in hippocampal mossy fiber sprouting. Here we have used slice cultures of hippocampus, in which mossy fibers sprout and form a supragranular plexus in response to granule cell deafferentation, and have compared cultures from early postnatal BDNF-deficient mice and wild-type mice. We demonstrate that there is sprouting of supragranular mossy fibers in cultured slices from both BDNF knock-out and wild-type mice. We conclude that BDNF is not an essential factor for mossy fiber sprouting. However, our data do not exclude a role for BDNF in mossy fiber sprouting in wild-type mice, as compensatory mechanisms might have become effective in the mutant.

Developmental distribution of a reeler gene-related antigen in the rat hippocampal formation visualized by CR-50 immunocytochemistry.

During histogenesis of the neocortex, Cajal Retzius cells in the marginal zone express the glycoprotein reelin which is developmentally regulated and involved in the formation of the inside out mode of cortical layering. Cajal Retzius cells are also present in the developing hippocampus. There, inhibition of reelin by blocking with CR-50, an antibody which recognizes the N-terminus of this protein, leads to abnormal development of layer-specific connections. Here we report the developmental distribution pattern of reelin expressing neurons in the rat hippocampal formation using CR-50 immunocytochemistry. Labelled Cajal Retzius cells were located near the hippocampal fissure in neonate rats. Many of these cells were still present in the adult. From postnatal day 4 on, neurons in other layers were stained with the CR-50 antibody. In adult rats immunopositive neurons were found in all hippocampal subfields and in the entorhinal cortex. These observations indicate that in the rat hippocampal formation reelin is expressed in different neuronal types during development and in adulthood. Moreover, Cajal Retzius cells in the marginal zone near the hippocampal fissure are still found in adult animals.

Sprouting in the hippocampus is layer-specific.

Partial removal of layer-specific afferents of the hippocampus is said to induce sprouting of intact fibers from neighboring layers that invade the zone of the degenerating axons. However, recent in vivo and in vitro studies using sensitive anterograde tracers have failed to demonstrate sprouting across laminar boundaries. Sprouting does occur; but, it mainly involves unlesioned fiber systems terminating within the layer of fiber degeneration in addition to the degenerating afferents. These findings point to rigid laminar cues attracting certain fiber systems while repelling others in normal development and after partial deafferentation.

A role for Cajal-Retzius cells and reelin in the development of hippocampal connections.

During development of the nervous system, specific recognition molecules provide the cues necessary for the formation of neural connections. In some regions, guiding cues for axonal pathfinding and target selection are provided by specific cells that exist only transiently during development, such as the floorplate or the cortical subplate. In the hippocampus, distinct groups of fibres innervate different layers. We have tested the hypothesis that transient neurons in the hippocampus provide positional information for the targeting of these fibres. Here we report that ablation of Cajal-Retzius cells in organotypic slice cultures of hippocampus prevented the ingrowth of entorhinal but not of commissural afferents. Experiments inhibiting Reelin (an extracellular matrix protein expressed by Cajal-Retzius cells) and analysis of reeler mutant mice showed dramatic abnormalities in the development of entorhinal afferents. Thus Cajal-Retzius cells and reelin are essential for the formation of layer-specific hippocampal connections.