Cholinergic fiber growth in co-cultures of CNS tissue.

In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells. To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity. Under these conditions, considerably fewer cholinergic cells appeared to survive. A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures. Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures. These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue. To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum. In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue. This failure might in part be related to the lack of trophic factors released by the target tissue. We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF. Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyaluronan-associated adhesive cues control fiber segregation in the hippocampus

In various brain regions, particularly in the hippocampus, afferent fiber projections terminate in specific layers. Little is known about the molecular cues governing this laminar specificity. To this end we have recently shown that the innervation pattern of entorhinal fibers to the hippocampus is mimicked by the lamina-specific adhesion of entorhinal cells on living hippocampal slices, suggesting a role of adhesion molecules in the positioning of entorhinal fibers. Here, we have analyzed the role of extracellular matrix components in mediating this lamina-specific adhesion. We show that hyaluronidase treatment of hippocampal slices abolishes lamina-specific adhesion as well as layer-specific growth of entorhinal fibers to the dentate outer molecular layer in organotypic slice cultures. We conclude that hyaluronan-associated molecules play a crucial role in the formation of the lamina-specific entorhinal projection to the hippocampus.     

Reformation of the severed septohippocampal cholinergic pathway in the adult rat by transplanted septal neurons

Summary Transplants containing developing cholinergic neurons were obtained from the septum-diagonal band area of rat fetuses and were implanted into a lesion of the septohippocampal cholinergic pathway or into a cavity of the occipital cortex in adult recipient rats. The growth of new cholinergic fibres from the implant into the hippocampal formation was followed with choline acetyltransferase (ChAT) determinations and acetylcholine esterase (AChE) histochemistry. A fimbrial lesion alone, transecting the septohippocampal pathway, caused an almost complete cholinergic denervation of the hippocampal formation that persisted throughout the five month experimental period. A septal transplant implanted into the cavity of the fimbrial lesion restored a new AChE-positive innervation pattern in the hippocampus and the dentate gyrus that closely mimicked the original innervation removed by the lesion. In parallel, there was a progressive recovery in the ChAT levels, starting in the septal end, and progressing in a temporal direction. A new cholinergic fibre supply could be established in the hippocampal formation also along an abnormal route, i.e. from the transplants implanted into a cavity in the occipital cortex (involving also the dorsal part of the entorhinal cortex). Provided the hippocampus previously had been denervated of its normal cholinergic innervation, a partly normal AChE-positive terminal pattern was thus re-established also from this abnormal position. If, on the other hand, the cholinergic afferents were left intact, the ingrowing fibres were restricted mainly to the outer portion of the dentate molecular layer, i.e. the terminal zone of the lesioned entorhinal perforant path fibres. This suggests that the growth of the sprouting AChE-positive fibres into the normal cholinergic terminal fields was blocked by the presence of an intact cholinergic innervation. It is concluded that regrowing cholinergic axons can be guided over large distances within the hippocampal formation, and that their patterning within the terminal fields is very precisely regulated by mechanisms released by deafferentation.     

The alvear pathway of the rat hippocampus

Neurons of the entorhinal cortex project to the hippocampus proper and dentate gyrus. This projection is called the ”perforant pathway” because it perforates the subiculum; current usage applies this term to all entorhino-hippocampal fibers. However, entorhinal fibers also reach Ammon’s horn via the alveus (”alvear pathway”), an alternative route first described by Cajal. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) was used in order to analyze the contribution of this pathway to the temporo-ammonic projection. In the temporal portion of the rat hippocampus, most of the entorhinal fibers reach Ammon’s horn after perforating the subiculum (classical perforant pathway). At more septal levels, the number of entorhinal fibers that take the alvear pathway increases; in the septal portion of the hippocampal formation, most of the entorhinal fibers to hippocampal subfield CA1 reach this subfield via the alveus. These fibers make sharp right-angle turns in the alveus, perforate the pyramidal cell layer, and finally terminate in the stratum lacunosum-moleculare. The crossed temporo-ammonic fibers reach their termination area in the stratum lacunosum-moleculare of CA1 almost exclusively via the alveus. These data indicate that the alveus is a major route by which entorhinal fibers reach their targets in CA1. Received: 14 May 1996 / Accepted: 22 June 1996     

Target- and maturation-specific membrane-associated molecules determine the ingrowth of entorhinal fibers into the hippocampus.

In this study the role of membrane-associated molecules involved in entorhinohippocampal pathfinding was examined. First outgrowth preferences of entorhinal neurites were analyzed on membrane carpets obtained from their proper target area, the hippocampus, and compared to preferences on control membranes from brain regions which do not receive afferent connections from the entorhinal cortex. On a substrate consisting of alternating lanes of hippocampal and control membranes, entorhinal neurites exhibited a strong tendency to grow on lanes of hippocampal membrane. These tissue-specific outgrowth preferences were maintained even on membrane preparations from adult brain tissue devoid of myelin. To determine the possible maturation dependence of these membranes, we examined guidance preferences of entorhinal neurites on hippocampal membranes of different developmental stages ranging from embryonic to postnatal and adult. Given a choice between alternating lanes of embryonic (E15-E16) and neonatal (P0-P1) hippocampal membranes, entorhinal neurites preferred to extend on neonatal membranes. No outgrowth preferences were observed on membranes obtained between E19 and P10. From P10 onward there was a reoccurrence of a preference for postnatal membrane lanes when neurites were presented with a choice between P15, P30, and adult membranes (>P60). This choice behavior of entorhinal neurites temporally correlates with the ingrowth of the perforant path into the hippocampus and with the stabilization of this brain area in vivo. Experiments in which postnatal and adult hippocampal membranes were heat inactivated or treated to remove molecules sensitive to phosphatidylinositol-specific phospholipase C demonstrated that entorhinal fiber preferences were controlled in this assay by attractive guidance cues and were independent of phosphatidylinositol-sensitive linked molecules. Moreover, entorhinal neurites displayed a positive discrimination for membrane-associated guidance cues of their target field, thus preferring to grow on membranes from the molecular layer of the dentate gyrus compared with CA3 or hilus membranes. Heat-inactivation experiments indicated that preferential growth of entorhinal axons is due to a specific attractivity of the molecular layer substrate. The data presented demonstrate that outgrowth of entorhinal fibers on hippocampal membranes is target and maturation dependent.     

Nerve growth factor expression in the developing hippocampus isolated in vitro.

Nerve growth factor (NGF) is synthesized in the hippocampus and neocortex and provides trophic support for afferent cholinergic neurons of the basal forebrain. To determine the capacity of the developing hippocampus to express NGF in the absence of NGF-responsive afferents, embryonic hippocampal cells isolated prior to septal innervation were studied in reaggregating cell culture. The expression of NGF protein in vitro was qualitatively and quantitatively similar to that observed in situ. The expression of NGF mRNA exhibited an initial increase in vitro but then plateaued and was maintained at a steady level. This latter finding was in contrast to the steady rise in NGF mRNA levels observed in situ. These data suggest that (i) intrinsic hippocampal interactions regulate the onset of NGF expression, but that (ii) additional extrinsic developmental signals may be required for proper regulation of hippocampal NGF expression during ontogeny.     

mRNA distribution within dendrites: Relationship to afferent innervation

The majority of neuronal mRNAs are confined to cell bodies, but a few mRNAs are present at high levels in dendrites. Here we report an initial analysis of the relationship between afferent innervation and the distribution of mRNA within dendritic fields. In situ hybridization techniques were used to compare the subcellular distribution of dendritic mRNAs in principal neurons of the hippocampal formation in vivo. The mRNA encoding the α subunit of calcium/calmodulin dependent protein kinase II (CAMII kinase) was present at high levels throughout the layers that contain the dendrites of hippocampal pyramidal cells and dentate granule cells. In contrast, the mRNA encoding the high molecular weight microtubule-associated protein MAP2 had a more limited distribution. In the dentate gyrus, labeling for MAP2 was present in a discrete band in the lamina containing proximal dendrites and decreased to low levels in laminae containing distal dendrites. This laminar pattern resembles the distinct terminations of the commissural/associational projection (high MAP2 labeling) and the entorhinal projection (lower MAP2 labeling) upon dendrites of granule cells. To determine if the differential distribution of dendritic mRNAs was regulated by either the presence or activity of afferents, we evaluated mRNA distribution in the dentate molecular layer following (1) removal of the entorhinal input by lesions of the entorhinal cortex or (2) prolonged delivery of potentiating stimulation to entorhinal afferents. Denervation led to modest decreases in the levels of mRNAs for both CAMII and MAP2 but did not lead to detectable alterations in mRNA distribution. Also, prolonged stimulation did not lead to detectable alterations in MAP2 or CAMII mRNA distribution, although such stimulation clearly elevated the expression of mRNA for glial fibrillary acidic protein (GFAP). © 1995 John Wiley & Sons, Inc.     

Specificity of a target cell-derived stop signal for afferent axonal growth

With a novel model culture system in which afferents are co-cultured with purified populations of target neurons, we have demonstrated that a target cell within the central nervous system (CNS), the cerebellar granule neuron, poses a ?stop-growing signal”? for its appropriate afferents, the mossy fibers. To ask whether this stop signal is afferent specific, we co-cultured granule neurons with an other cerebellar afferent system, the climbing fibers from the inferior olivary nuclei, which normally contact Purkinje neurons, and with retinal ganglion cell afferents, which never enter the cerebellum. Granule neurons do not pose a stop signal to either of these afferents. In contrast to pontine mossy afferents that grow well on laminin and showed reduced outgrowth on granule neurons, both olivary and retinal fibers displayed similar growth on laminin alone or on granule neurons. In addition, each afferent showed different degrees of fasciculation and growth cone morphology on laminin. Thus, the growth arrest signal sent by granule neurons is specifically recognized by their appropriate afferents. Moreover, these three types of afferents exhibit varying growth patterns on the same noncellular and cellular substrates, implicating distinct molecular characteristics of growth regulation for different classes of neurons that would contribute to specificity of synapse formation. © 1992 John Wiley & Sons, Inc.     

Specificity of a target cell-derived stop signal for afferent axonal growth.

With a novel model culture system in which afferents are co-cultured with purified populations of target neurons, we have demonstrated that a target cell within the central nervous system (CNS), the cerebellar granule neuron, poses a "stop-growing signal" for its appropriate afferents, the mossy fibers. To ask whether this stop signal is afferent specific, we co-cultured granule neurons with another cerebellar afferent system, the climbing fibers from the inferior olivary nuclei, which normally contact Purkinje neurons, and with retinal ganglion cell afferents, which never enter the cerebellum. Granule neurons do not pose a stop signal to either of these afferents. In contrast to pontine mossy afferents that grow well on laminin and showed reduced outgrowth on granule neurons, both olivary and retinal fibers displayed similar growth on laminin alone or on granule neurons. In addition, each afferent showed different degrees of fasciculation and growth cone morphology on laminin. Thus, the growth arrest signal sent by granule neurons is specifically recognized by their appropriate afferents. Moreover, these three types of afferents exhibit varying growth patterns on the same noncellular and cellular substrates, implicating distinct molecular characteristics of growth regulation for different classes of neurons that would contribute to specificity of synapse formation.     

Restoration of High Affinity Choline Uptake in the Hippocampal Formation Following Septal Cell Suspension Transplants in Rats with Fimbria-Fornix Lesions

High affinity choline uptake (HACU) was investigated in the hippocampal formation following fetal septal cell suspension transplants into rats with fimbria-fornix lesions. Nine-14 weeks after transplantation, HACU was markedly decreased in hippocampi from animals with fimbria-fornix lesions; this decrease was ameliorated by fetal septal cells transplanted into the host hippocampus. HACU related to septal transplantation was activated in vitro by K+, and in vivo by the administration of scopolamine and picrotoxin. These findings suggest that fetal septal cell transplantation can restore HACU in the host hippocampus following fimbria-fornix lesions, and that HACU related to the graft has pharmacological properties similar to those of the normal adult HACU system. The activation of HACU by picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, suggests that transplanted cholinergic neurons receive either direct or indirect functional input from GABAergic afferents from the transplant and/or host hippocampus. Lesions of the fimbria-fornix also resulted in an increased binding to muscarinic receptors in the dorsal hippocampus. This increase in binding was not significantly ameliorated by intrahippocampal grafts of cholinergic neurons.     

Interactions between entorhinal axons and target hippocampal neurons: a role for glutamate in the development of hippocampal circuitry

A coculture system consisting of input axons from entorhinal cortex explants and target hippocampal pyramidal neurons was used to demonstrate that glutamate, released spontaneously from afferent axons, can influence both dendritic geometry of target neurons and formation of presumptive synaptic sites. Dendritic outgrowth was reduced in hippocampal neurons growing on entorhinal axons when compared with neurons growing off the axons. Presumptive presynaptic sites were observed in association with hippocampal neuron dendrites and somas. HPLC analysis showed that glutamate was released from the explants in an activity- and Ca2(+)-dependent manner. The general glutamate receptor antagonist D-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca2+ also promoted dendritic outgrowth and reduced the formation of presumptive synaptic sites. The results suggest that the neurotransmitter glutamate may play important roles in the development of hippocampal circuitry.     

Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetylcholinesterase activity

The postnatal development of acetylcholinesterase (AChE) activity in the hippocampal formation of the developing rat brain, as demonstrated histochemically by the copper-thiocholine technique, serves as a marker for the ingrowing cholinergic afferent fibers. The discrete laminar pattern of staining characteristic of the adult hippocampal formation develops entirely after birth. Stain deposit is observable earliest (about 4 days after birth) at the septal end of the hippocampus. During the following week, AChE activity can be demonstrated in successively temporal segments until, about 11 days after birth, all parts of the hippocampal formation exhibit activity. Within each segment, the pattern of developing activity suggests association with three distinct fiber projections emanating from the fimbria, each with its own characteristic time of appearance and rate of growth: (1) a projection through stratum oriens of hippocampus regio inferior to stratum oriens of regio superior; (2) fibers which cross straum pyramidale of regio inferior, run in the suprapyramidal zone of that region and continue into the supra- and infragranular zones in the external leaf of the dentate gyrus; (3) a projection through stratum oriens of regio inferior which continues into the supra- and infragranular zones in the internal leaf of the dentate gyrus.     

BIOCHEMICAL CORRELATES OF TRANSMISSION MEDIATED BY GLUTAMATE AND ASPARTATE

Abstract— Glutamate and aspartate probably serve as transmitters of hippocampal perforant path and commissural afferents, respectively. We therefore used slices of hippocampal regions to evaluate certain biochemical properties as markers for sites of transmission mediated by these amino acids. In these studies content and accumulation of glutamate and aspartate were compared with their Ca²⁺-dependent effluxes.
Hippocampal regions varied little in their contents of glutamate and aspartate, but slices of regio superior and dentate gyrus accumulated and released more of each than slices of regio inferior. A commissurotomy or bilateral entorhinal lesion altered Ca²⁺-dependent efflux and accumulation in the same direction, but did not affect the glutamate or aspartate content of any hippocampal region. Elimination of hippocampal mossy fibers reduced the Ca²⁺-dependent efflux of glutamate and probably aspartate from slices of dentate gyrus, but not of regio inferior, where most mossy fiber synapses are located. The mossy fibers appeared relatively deficient in aspartate in both strains tested, but only in Purdue-Wistar rats were they enriched in glutamate. Removal of the perforant path input to the fascia dentata did not significantly change the activity of any of the enzymes most actively involved in glutamate synthesis.
These results suggest that accumulation or high affinity transport of glutamate or aspartate can be employed to localize afferents which use these amino acids as transmitters, although it is not so reliable or selective a marker as Ca²⁺-dependent efflux. Enrichment in either glutamate or aspartate content or in the activity of enzymes which synthesize them is not a reliable marker. Neither amino acid is likely to be used as a transmitter by the hippocampal mossy fibers.     

Quantitative analysis of the distribution of acetylcholinesterase-positive senile plaques in the hippocampal formation in patients with senile dementia of Alzheimer type

Senile plaques (SP) are one of the major neuropathologic hallmarks of senile dementia of Alzheimer type (SDAT). The regional distribution of SP stained for acetylcholinesterase (AChE) has been quantified in the hippocampal formation of patients with SDAT. Consistent differences in plaque distribution and density were observed between different patients. SP were significantly more numerous in the outer dentate molecular layer, whereas their density was low in other hippocampal regions. The quantity of AChE positive SP seemed to correlate with the density of neurofibrillary tangles in the entorhinal cortex. The AChE positive SP had a pattern of distribution different from the one observed with Thioflavin S. These results are discussed in light of a possible sprouting of cholinergic septal afferents.     

Entorhinal denervation induces homeostatic synaptic scaling of excitatory postsynapses of dentate granule cells in mouse organotypic slice cultures

Denervation-induced changes in excitatory synaptic strength were studied following entorhinal deafferentation of hippocampal granule cells in mature (≥ 3 weeks old) mouse organotypic entorhino-hippocampal slice cultures. Whole-cell patch-clamp recordings revealed an increase in excitatory synaptic strength in response to denervation during the first week after denervation. By the end of the second week synaptic strength had returned to baseline. Because these adaptations occurred in response to the loss of excitatory afferents, they appeared to be in line with a homeostatic adjustment of excitatory synaptic strength. To test whether denervation-induced changes in synaptic strength exploit similar mechanisms as homeostatic synaptic scaling following pharmacological activity blockade, we treated denervated cultures at 2 days post lesion for 2 days with tetrodotoxin. In these cultures, the effects of denervation and activity blockade were not additive, suggesting that similar mechanisms are involved. Finally, we investigated whether entorhinal denervation, which removes afferents from the distal dendrites of granule cells while leaving the associational afferents to the proximal dendrites of granule cells intact, results in a global or a local up-scaling of granule cell synapses. By using computational modeling and local electrical stimulations in Strontium (Sr(2+))-containing bath solution, we found evidence for a lamina-specific increase in excitatory synaptic strength in the denervated outer molecular layer at 3-4 days post lesion. Taken together, our data show that entorhinal denervation results in homeostatic functional changes of excitatory postsynapses of denervated dentate granule cells in vitro.     

A membrane glycoprotein associated with the limbic system mediates the formation of the septo-hippocampal pathway in vitro

The ability of a neuronal surface glycoprotein to mediate the formation of neuronal connections was tested in an explant culture system. A monoclonal antibody against the limbic system-associated membrane protein (LAMP) was used in co-cultures containing cholinergic neurons of the septum and their hippocampal target neurons. Antibody treatment had no effect on general axon outgrowth, but significantly diminished the ability of septal cholinergic axons to invade and collateralize in the hippocampus. The results suggest that factors regulating general axon outgrowth may be distinct from those regulating the patterns of outgrowth that define the formation of neural circuits.     

Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.     

Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells

Purkinje cells are the principal neurons of the cerebellar cortex and are characterized by a large and highly branched dendritic tree. For this reason, they have for a long time been an attractive model system to study the regulation of dendritic growth and differentiation. In this article, I will first review studies on different aspects of Purkinje cell dendritic development and then go on to present studies which have aimed at experimentally altering Purkinje cell dendritic development. Some of the cellular and molecular mechanisms which have been shown by these studies to be important determinants of Purkinje cell dendritic development will be discussed, in particular the role of the parallel fiber input, of hormones, and of neuronal growth factors. The organotypic slice culture method will be introduced as an important experimental tool to study Purkinje cell dendritic development under controlled conditions. Using cerebellar slice cultures, protein kinase C (PKC) has been identified as a major determinant of Purkinje cell dendritic development and the contribution of specific isoforms of PKC will be discussed. Finally, it will be shown that Purkinje cell dendritic development in slice cultures does not depend on the activation of glutamate receptors and appears to be independent of the presence of the neurotrophin BDNF. These studies indicate that the initial outgrowth of the Purkinje cell dendritic tree can occur in the absence of signals derived from afferent fibers, but is under control of PKC signaling.