A comparative study of prenatal development in the olfactory bulb, neocortex and hippocampal region of the precocial mouse Acomys cahirinus and rat

Unlike the remainder of the rodent subfamily Muridae, Acomys cahirinus (the 'spiny' mouse) is born in a precocial state after a long (39 day) gestation. In this paper, the development of the olfactory bulb, neocortex and hippocampal formation of Acomys from prenatal days 14-34 was examined and the rate of maturation compared with that of its cousin, the laboratory rat (Rattus norvegicus). At the earliest stages examined, Acomys was approximately 2 days less mature than the same post-conception aged rat. The difference between the two species increased: Acomys at 28 days postconception resembled the 22-day rat. By the end of gestation, Acomys and the rat were in a relatively similar developmental state. Therefore, Acomys exhibits a quite different timetable of early maturation which includes a protracted period of relatively slow growth during mid-gestation. As such, it offers many benefits as a subject for studies of both early ontogenesis and the mechanisms which result in species differences.     

The development of the dentate area and the hippocampal mossy fiber projection of the rat

The development of the dentate area and the hippocampal mossy fiber system of the rat has been investigated at the light microscopic level by using fluorescent tracing, Nissl, and Timm's histochemical methods. Although the cytoarchitectonic development of the dentate granular layer is mainly a postnatal phenomenon, the initial events take place before birth. The aggregation and maturation of the cells in the granular layer proceed in a graded fashion from the lateral to the medial and from the superficial to the deep aspects of the layer. The earliest-formed granule cells are probably derived directly from the cells of the ventricular zone. They start to form mossy fibers prenatally, either during the relatively long period of migration to the granular layer or soon after their arrival. However, most of the granule cells are derived from a secondary proliferative center in the hilus. They start to produce mossy fibers postnatally a while after arriving at the granular layer. The total complement of granule cells starts to grow mossy fibers in a sequence that is related to the final position of the cells of origin within the granular layer. This sequence also proceeds in a graded fashion from the lateral to the medial and from the superficial to the deep aspects of the layer. In the beginning the mossy fibers elongate relatively rapidly. Already at birth the Timm-stained mossy fiber zone occupies the anterolateral part of the hilus and the adjacent suprapyramidal parts of the regio inferior. Once the mossy fibers have reached the distal end of the regio inferior they elongate along the longitudinal axis of the hippocampus more slowly. At the same time the Timm-stainability of the mossy fiber zone, which, during the first postnatal week, is weaker toward the regio superior, develops a mature pattern in which the distal part of the zone stains most intensely. Throughout development, fibers from the granule cells that form first are longer and diverge more in the septotemporal dimension than fibers from later-forming granule cells. In contrast to other axonal systems which appear to be sculptured from a diffuse set of connections the results presented here provide evidence that the topographic relationships of the mossy fiber system develop in a stepwise fashion.     

Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: Visualization after retrograde transport of biocytin

In complex partial epilepsy and in animal models of epilepsy, hippocampal mossy fibers appear to develop recurrent collaterals, that invade the dentate molecular layer. Mossy fiber collaterals have been proposed to subserve recurrent excitation by forming granule cell-granule cell synapses. This hypothesis was tested by visualizing dentate granule cells and their mossy fibers after terminal uptake and retrograde transport of biocytin. Labeling studies were performed with transverse slices of the caudal rat hippocampal formation prepared 2.6–l70.0 weeks after pilocarpine-induced or kainic acid-induced status epilepticus. Light microscopy demonstrated the progressive growth of recurrent mossy fibers into the molecular layer; the densest innervation was observed in slices from pilocarpine-treated rats that had survived 10 weeks or longer after status epilepticus. Thin mossy fiber collaterals originated predominantly from deep within the hilar region, crossed the granule cell body layer, and formed an axonal plexus oriented parallel to the cell body layer within the inner one-third of the molecular layer. When sprouting was most robust, some recurrent mossy fibers at the apex of the dentate gyrus reached the outer two-thirds of the molecular layer. The distribution and density of mossy fiber-like Timm staining correlated with the biocytin labeling. When viewed with the electron microscope, the inner one-third of the dentate molecular layer contained numerous mossy fiber boutons. In some instances, biocytin-labeled mossy fiber boutons were engaged in synaptic contact with biocytin-labeled granule cell dendrites. Granule cell dendrites did not develop large complex spines (“thorny excrescences”) at the site of synapse formation, and they did not appear to have been permanently damaged by seizure activity. These results establish the validity of Timm staining as a marker for mossy fiber sprouting and support the view that status epilepticus provokes the formation of a novel recurrent excitatory circuit in the dentate gyrus. Retrograde labeling with biocytin showed that the recurrent mossy fiber projection often occupies a considerably greater fraction of the dendritic region than previous studies had suggested. © 1995 Wiley-Liss, Inc.     

A stereological study of neocortical maturation in the precocial mouse, Acomys cahirinus

The development of neocortical area 17 of Acomys cahirinus was studied by quantifying changes in the numerical density of neurons and glia, and by examinations of subjects treated on the day of birth with tritiated thymidine. Unlike most laboratory species, Acomys is born in a relatively advanced state, with open and functional ears and eyes and coordinated locomotor abilities. Rapid neocortical growth occurred during the first 60 postnatal days and was accompanied by a 65% decrease in neuronal packing density. No evidence of neuronal proliferation was found in the neocortex, although labelling was found in the dentate gyrus and olfactory bulb. Glial densities remained relatively constant through the same time period, a consequence of continued proliferation. While previous studies have demonstrated that Acomys and the laboratory mouse undergo quite different patterns of olfactory bulb and hippocampal formation growth, the present study indicates that patterns of neocortical maturation are very similar in the two species. These findings suggest large variations both within and between species in patterns of brain growth.     

The organization and development of the hippocampal mossy fiber system

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat.The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus.During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.     

Hippocampal sympathetic ingrowth in rats and guinea pigs: quantitative morphometry and topographical differences

Sympathetic ingrowth is an unusual neural rearrangement in response to damage of the septohippocampal pathway in which peripheral noradrenergic nerves grow into the hippocampal formation. Hippocampal ingrowth has been extensively studied in rats and has been suggested to be regulated by the mossy fibers of the dentate granule cells, hippocampal interneurons, or glial cells. Sympathetic ingrowth was found to occur in both rats and guinea pigs; however, a discrepancy between the species was observed in the topographical distribution of sympathetic ingrowth. Ingrowth fibers were found in the dentate hilus and area CA3 of guinea pigs and rats. However, in the guinea pig fibers extended into area CA1. Quantitative estimates of fiber number confirmed these observations and identified significant differences between the species in the intrahippocampal lamellar distribution of ingrowth fibers. The topographical differences in sympathetic ingrowth could not be explained by differences in the distribution of the mossy fibers (Timms stain), cholinergic septal afferents (anterograde HRP), or in hippocampal interneurons (GAD-immunoreactive neurons). These species differences are challenging to current theories concerning the regulation of sympathetic ingrowth and may provide a useful model for testing further hypothesis about axonal guidance and target selection.     

Postnatal development and synaptic connections of hilar mossy cells in the hippocampal dentate gyrus of rhesus monkeys

Mossy cells of the hippocampal dentate gyrus were analyzed through postnatal development. At birth, a few thorny excrescences were found on the proximal dendrites of mossy cells, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number and complexity until the third month. After that age, the complexity of thorny excrescences is so great that an increase in spine density can be seen only in electron microscopic preparations. An increase in the number of pedunculate spines per unit length of distal dendrite was detected via light microscopy during the first 9 postnatal months. The somata and dendrites of mossy cells displayed adult-like characteristics after the ninth postnatal month. Mossy fiber terminals at birth frequently displayed immature ultrastructural characteristies and formed synapses with dendritic shafts and spines. At later postnatal ages and in adults, axospinous synapses were found almost exclusively. This is consistent with the postnatal development of the complex spines of the mossy cells. Axons of mossy cells were generally confined to the hilus in our 150 -μm-thick sections, where they gave rise to several collaterals. The axon terminals from these collaterals formed asymmetric synapses with dendrites and dendritic spines in the hilar region of the dentate gyrus. These data provide the first anatomical evidence that hilar mossy cells of the primate dentate gyrus have excitatory projections similar to their equivalent cell type in subprimates. The present study indicates that mossy cells of the dentate gyrus are in a more advanced stage of development at birth and mature faster than similar neurons of the human hippocampus. This may represent a faster maturation of hippocampal circuitry in nonhuman primates compared to that in the human.     

Postnatal development of CA3 pyramidal neurons and their afferents in the Ammon's horn of rhesus monkeys

Previous studies described the postnatal development of CA3 pyramidal neurons and their afferents in the rat. However, the postnatal development of the primate hippocampus was not previously studied. Thus, pyramidal neurons of the CA3 area of the monkey hippocampus were analyzed postnatally in the present study. At birth, a few thorny excrescences, the complex spines postsynaptic to mossy fibers, were found on the proximal segments of both apical and basal dendrites, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number until the third month. A continuous increase in the number of spines per unit length along the distal dendrites was observed during the first 12 months. The ultrastructural features of somata and dendrites of pyramidal cells in newborn monkeys were similar to those of adults. The analysis of the afferents to the CA3 pyramidal neurons was limited to the development of mossy fibers, the axons of granule cells, and myelinated axons in the alveus, stratum oriens, and stratum lacunosum-moleculare. At birth, most mossy fiber terminals were densely packed with synaptic vesicles and formed mainly axospinous synapses with CA3 pyramidal cells. By 1 month of age, the number of mitochondria and embedded spines increased to mature amounts. In the first postnatal month, degenerating axons and axon terminals were frequently observed in the mossy fiber bundles in stratum lucidum. The proportion of myelinated axons increased simultaneously in all three examined layers. At birth most axons were unmyelinated, whereas at 7 months of age the proportion of myelinated axons was similar to that found in adults. The present study indicates that most pyramidal neurons of the CA3 region in monkeys are in an advanced stage of development at the time of birth. Thus, mossy fibers from granule cells in the dentate gyrus have established mature-looking synapses, and the thorny excrescences of pyramidal cells that are postsynaptic to mossy fibers are also adult-like. Nevertheless, several of the adult features, such as the spine density of distal dendrites of pyramidal neurons and the myelination of afferent axons, develop during an extended period of time in the first year. The significance of this early anatomical maturation in a brain region involved in memory function is consistent with recent behavioral data that show a rapid postnatal maturation of limbic-dependent recognition memory in rhesus monkeys. © 1995 Wiley-Liss, Inc.     

Comparative localization of enkephalin and cholecystokinin immunoreactivities and heavy metals in the hippocampus

The present study is concerned with the cellular origins and identities of the hippocampal enkephalin and CCK-immunoreactive fibers and terminals. In the hippocampus of the rat, the guinea pig and the European hedgehog a system of enkephalin immunoreactive nerves emerges in the hilus of area dentata and can be followed to the apical dendrites of the hippocampal regio inferior pyramidal cells. This pattern of immunoreactive nerves corresponds to the hippocampal mossy fiber system as visualized by the Timm staining. Cholecystokinin immunoreactive nerve fibers and terminals reveal the same distribution in the guinea pig and the European hedgehog whereas in the rat the mossy fiber zone contains little or no cholecystokinin immunoreactivity. In the guinea pig degeneration of the mossy fibers after stereotactic lesions of the mossy fibers causes a complete loss of both enkephalin and cholecystokinin immunoreactivity in the mossy fiber zone. Only a few enkephalin immunoreactive cell bodies were scattered throughout the granular cell layer of area dentata, but inhibition of the axoplasmic transport by colchicine dramatically increased the number of enkephalin immunoreactive granule cell bodies. Enkephalin immunoreactive cell bodies were also detected in the hilus, throughout the pyramidal cell layer as well as in the stratum radiatum and stratum moleculare. Cholecystokinin immunoreactive cell bodies were seen in the hilus of the area dentata and in the stratum oriens, stratum pyramidale and stratum radiatum and the cell-rich layer of subiculum. No cholecystokinin immunoreactive cell bodies were observed in the granular cell layer of area dentata. Even after colchicine treatment the granule cells were devoid of cholecystokinin immunoreactivity. In the rat a system of nerves displaying enkephalin immunoreactivity was also observed in the superficial one-third of the molecular layer of area dentata, a zone which corresponds to the termination of the lateral perforant path. Another observation was that in the rat, but not in the guinea pig and the hedgehog, the terminal zone of both the medial perforant path and the zone of commissural and associational fibers of area dentata contained cholecystokinin immunoreactive molecules. In summary, our data show: (1) that the hippocampal mossy fibers contain enkephalin immunoreactive molecules; (2) that the cholecystokinin immunoreactivity in the mossy fiber zone is most likely also localized in the mossy fibers per se, although the granule cells seem devoid of cholecystokinin immunoreactivity; (3) zinc, here visualized as a Timm-positive substance, is also localized in the mossy fiber terminals; further, (4) other intrinsic cell bodies than the granule cells may contribute to both the enkephalin and cholecystokinin immunoreactive terminals within the hippocampus; (5) in the rat the lateral perforant path may be enkephalinergic; and (6) both the terminal zone of the medial perforant path and the associational and commissural fibers of the rat contain cholecystokinin immunoreactivity.     

Growth of hippocampal mossy fibers: a lesion and coculture study of organotypic slice cultures

In hippocampal slice cultures, the mossy fibers from the dentate granule cells project as normally to cells in the dentate hilus (CA4) and the hippocampal CA3 pyramidal cells. After lesions in vivo and intracerebral transplantation, the mossy fibers can alter their normal distribution within CA3 and even contact CA1 pyramidal cells. The present study examined whether similar changes could be induced in the more simple, virtual two-dimensionally organized slice cultures. For this purpose slices of 7-day-old hippocampi were prepared and subjected to one of the two following manipulations: (1) transection of the mossy fiber layer in CA3 or (2) rearrangement of the geometrical relations between the dentate granule cells in their potential targets (CA3 and CA1) by coculturing dentate slices with CA3 or CA1 slices. Two to 8 weeks later the distribution of the mossy fiber system was visualized by histochemical Timm sulphide silver staining of the terminals. The distribution of the mossy fiber system observed in previous studies of ordinary hippocampal slice cultures was confirmed. In addition, mossy fibers were found to cross the cuts through the mossy fiber layer with formation of a reduced number of characteristic Timm-stained terminals in CA3 distal to the lesion. Close proximity and contiguity of the cut surfaces were important for such growth to occur. Significantly fewer mossy fiber terminals were found when separate slices of dentate and CA3 tissue were joined and grown as cocultures. Similar apposition of slices of dentate and CA1 tissue only rarely resulted in the ingrowth of mossy fibers into CA1. The Timm-stained mossy fiber terminals were then of subnormal size. The results show the potentials of the slice culture technique in supplementing lesion and transplant studies in situ. The growth of mossy fibers across a transection of their pathway is thus a new observation, difficult to demonstrate in the brain. The limited growth in the cocultures of aberrant mossy fibers into Ca1 does, on the other hand, emphasize the importance of close structural contact for the formation of nerve connections, and such contact is apparently more easily obtained in the brain. When the growth of the mossy fibers and that of the cholinergic septohippocampal fibers are compared, it is evident that the cholinergic axons grow better both in vitro and in vivo after lesions and transplantation.     

The hippocampal mossy fiber system of the rat studied with retrograde tracing techniques. Correlation between topographic organization and neurogenetic gradients

The topographical organization of the hippocampal mossy fiber system, which connects the dentate granule cells with the pyramidal cells of the regio inferior, has been examined in rats with retrograde tracing methods. Following the application of the fluorescent dye True Blue to different parts of the mossy fiber layer in the hippocampal regio inferior, retrogradely labeled granule cells were observed in the dentate fascia. The distribution of the labeled cells within the dentate granule cell layer indicates that all mossy fibers have an almost parallel, slightly descending course in regio inferior near the dentate hilus. In the ventricular part of regio inferior, particularly toward the transition to the regio superior, the mossy fibers are sorted out according to the position of their parent cell bodies within the granular layer. Near the transition to regio superior the fibers from lateral granule cells extend both septally and temporally over a longer distance than the fibers from more medial cells. Similarly, the fibers coming from the superficial cells extend both septally and temporally over a longer distance than those from the deep cells. The mossy fibers arising from a specific septotemporal level of the dentate fascia innervate a segment of the regio inferior that extends approximately 180 μm above to approximately 1,600 μm below the level of origin. Similar results were obtained following injections of Nuclear Yellow and horseradish peroxidase. Since previous studies have demonstrated that the granule cells are formed along gradients from lateral to medial and from superficial to deep, there appears to be a correlation between the formation, and hence the position, of the granule cells and the topography of their projection into the regio inferior.     

Maturation of cultured hippocampal slices results in increased excitability in granule cells

The preparation of hippocampal slices results in loss of input neurons to dentate granule cells, which leads to the reorganization of their axons, the mossy fibers, and alters their functional properties in long-term cultures, but its temporal aspects in the immature hippocampus are not known. In this study, we have focused on the early phase of this plastic reorganization process by analyzing granule cell function with field potential and whole cell recordings during the in vitro maturation of hippocampal slices (from 1 to 17 days in vitro, prepared from 6 to 7-day-old rats), and their morphology using extracellular biocytin labelling technique. Acute slices from postnatal 14-22-day-old rats were analyzed to detect any differences in the functional properties of granule cells in these two preparations. In field potential recordings, small synaptically-evoked responses were detected at 2 days in vitro, and their amplitude increased during the culture time. Whole cell voltage clamp recordings revealed intensive spontaneous excitatory postsynaptic currents, and the susceptibility to stimulus-evoked bursting increased with culture time. In acutely prepared slices, neither synaptically-evoked responses in field potential recordings nor any bursting in whole cell recordings were detected. The excitatory activity was under the inhibitory control of gamma-aminobutyric acid type A receptor. Extracellularily applied biocytin labelled dentate granule cells, and revealed sprouting and aberrant targeting of mossy fibers in cultured slices. Our results suggest that reorganization of granule cell axons takes place during the early in vitro maturation of hippocampal slices, and contributes to their increased excitatory activity resembling that in the epileptic hippocampus. Cultured immature hippocampal slices could thus serve as an additional in vitro model to elucidate mechanisms of synaptic plasticity and cellular reactivity in response to external damage in the developing hippocampus.     

Lesion-induced rerouting of hippocampal mossy fibers in developing but not in adult rats

The organization of the hippocampal mossy fiber system which projects from the granule cells of fascia dentata to the pyramidal cells of the hippocampal regio inferior (CA3) was studied after chronic lesions had been applied to CA3 in neonatal (0–3 day old), 5 and 21 day old, and adult rats. The distribution of terminals was monitored by the histochemical Timm sulphide silver method, and the fibers were demonstrated by the original Nauta silver method. Complete transections of CA3 in neonatal rats induced a layer of aberrant, infrapyramidal mossy fiber terminals in CA3 up to 1,300 μm septal to the transection followed by a corresponding expansion of the suprapyramidal mossy fiber layer for at least 720 μm. At increasing distance above the lesion, the induced changes moved laterally in CA3, affecting more distal parts of the mossy fibers. Corresponding to the layer of aberrant infrapyramidal terminals were aberrant bundles of mossy fibers, with an abnormally ascending septal course. No aberrant mossy fibers or terminals were induced temporal to the complete transections. Partial CA3 lesions in neonates induced changes corresponding to those observed after complete transections septal to the lesions. In addition, a layer of aberrant infrapyramidal mossy fiber terminals was induced in CA3 as far as 900 μm temporal to the partial lesions, accompained by an expansion of the suprapyramidal layer. The infrapyramidal terminals were related to aberrant bundles of fibers which followed an abnormally steep, temporal course, from which they in turn leveled off to join the suprapyramidal bundles. Compared to neonatal lesions, hippocampal transections performed on day 5 induced a slightly less dense layer of aberrant infrapyramidal mossy fiber terminals in CA3, and although a transection on day 21 still induced aberrant infrapyramidal terminals, these were far less abundant than after lesions at day 5. Transections in adult rats induced no such changes at all, and additional denervation of CA3 by simultaneous removal of the commisural projection from the contralateral hippocampus did not have any effect. The lesion-induced, but age-dependent redistribution of mossy fibers to CA3 is discussed in terms of reactive reinnervation following partial deafferentation, and compensatory sprouting and rerouting due to pruning-like effects of the lesions. A rerouting of developing, not fully matured mossy fibers is found to be most likely: Deprived of normal target areas, the fibers have been forced to grow in abnormal directions, ending up hyperinnervating adjacent, accessible levels through the formation of aberrant infrapyramidal terminals and expansion of the suprapyramidal zone.     

The organization and development of the hippocampal mossy fiber system

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat. The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus. During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.     

Consequences of neonatal seizures in the rat: Morphological and behavioral effects

Whereas neonatal seizures are a predictor of adverse neurological outcome, there is controversy regarding whether seizures simply reflect an underlying brain injury or can cause damage. We subjected neonatal rats to a series of 25 brief flurothyl-induced seizures. Once mature the rats were compared with control littermates for spatial learning and activity level. Short-term effects of recurrent seizures on hippocampal excitation were assessed by using the intact hippocampus formal preparation and long-term effects by assessing seizure threshold. Brains were analysed for neuronal loss, sprouting of granule cell axons (mossy fibers), and neurogenesis. Compared with controls, rats subjected to neonatal seizures had impaired learning and decreased activity levels. There were no differences in paired-pulse excitation or inhibition or duration of afterdischarges in the intact hippocampal preparation. However, when studied as adults, rats with recurrent flurothyl seizures had a significantly lower seizure threshold to pentylenetrazol than controls. Rats with recurrent seizures had greater numbers of dentate granule cells and more newly formed granule cells than the controls. Rats with recurrent seizures also had sprouting of mossy fibers in CA3 and the supragranular region. Recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development.     

Abnormal mGluR2/3 expression in the perforant path termination zones and mossy fibers of chronically epileptic rats

Epilepsy is characterized by hyperexcitability of hippocampal networks, excessive release of glutamate, and progressive neurodegeneration. Presynaptic group II metabotropic receptors (mGluR2 and mGluR3) are among different mechanisms that modulate presynaptic release of glutamate, especially at the mossy fibers in the hippocampus. Here, we explore whether mGluR2/3 expression is affected in a rat model of temporal lobe epilepsy obtained via pilocarpine-induced status epilepticus (SE). Immunohistochemical assays were performed in age-matched controls and two groups of epileptic rats sacrificed at 25-35 days (1 month post-SE) and at 55-65 days (2 months post-SE) following SE onset. A dramatic lessening of mGluR2/3 immunofluorescence was observed at CA1 and CA3 stratum lacunosum/molecular (SLM) declining to 60% and 68% of control values in 1-month and 2-month post-SE, respectively. Additionally, thickness of mGluR2/3-stained SLM layer narrowed up to 70% of controls indicating atrophy at this branch of the perforant path. Epileptic rats exhibited a marked and progressive down-regulation of mGluR2/3 expression in mossy fiber at hilus and CA3 stratum lucidum in contrast with an enhanced expression of vesicular glutamate transporter type 1 (VGluT1) at the mossy fibers. Intense VGluT1 punctated staining was detected at the inner third molecular layer indicating glutamatergic sprouting. In the molecular layer, mGluR2/3 labeling slightly declined in the 1-month post-SE group but then increased in the 2-month post-SE group although it was diffusely distributed. Down-regulation of mGluR2/3 at the mossy fibers and the SLM may render epileptic hippocampal networks hyperexcitable and susceptible to glutamate-mediated excitotoxicity and neurodegeneration.     

Postnatal maturation of brain cholinergic systems in the precocial murid Acomys cahirinus: Comparison with the altricial rat

The spiny mouse (Acomys cahirinus) is the only precocial murid species. It has some neuroanatomical peculiarities such as a relatively thin cerebral cortex and a large hippocampus. The levels of choline acetyltransferase, membrane-bound acetylcholinesterase and muscarinic receptor sites (measured as [3H]quinuclidynil benzilate binding) were assessed in the whole brain on days 1, 7, 14, 21, 28 and 80 (adult), and compared with those of Wistar rats of the corresponding ages. At birth choline acetyltransferase was significantly higher in spiny mice than in rats but the adult levels were similar, with an overall increase of about 5.2- and 14-fold for the former and the latter species, respectively. Membrane-bound acetylcholinesterase level and maximal density of muscarinic receptor sites in spiny mice were considerably higher at birth, in contrast adult levels were significantly lower than in rats with a respective overall increase of about 1.5- and over 4.5-fold. The high degree of maturity attained at birth by spiny mice partially depends on the long gestation period. However, if we consider postconception age, the maturation of choline acetyltransferase appears to be delayed at birth in the spiny mice, probably in relation to the lack of external stimulation during intrauterine life. In the cerebral cortex, hippocampus and striatum of adult spiny mice, when compared with the rats, there were similar levels of choline acetyltransferase but lower levels of membrane-bound acetylcholinesterase and, in the cerebral cortex, lower density of muscarinic receptor sites.     

Distal infrapyramidal and longitudinal mossy fibers at a midtemporal hippocampal level

Timm's and horseradish peroxidase histochemical methods were used to demonstrate a small, distal infrapyramidal mossy fiber projection at midtemporal hippocampal levels in the rat. The Timm's sulfide silver method revealed sparse granules of dark, mossy fiber-like staining at an infrapyramidal location in an area roughly equivalent to hippocampal subfield CA3a (close to the CA1 border). The use of anterograde transport of horseradish peroxidase enabled the visualization of short mossy fiber axons that left the suprapyramidal bundle, penetrated the pyramidal cell layer, and terminated in a distal infrapyramidal position. The HRP-labeled axons possessed periodic swellings that are characteristic of mossy fiber morphology. The HRP studies also provided evidence for longitudinally oriented mossy fibers at midtemporal hippocampal levels. The distal tip of the suprapyramidal band of mossy fibers turned temporally and could be detected up to 1140 micrometers ventral to the main suprapyramidal band.     

Characterization of Target Cells for Aberrant Mossy Fiber Collaterals in the Dentate Gyrus of Epileptic Rat

Previous studies have demonstrated formation of recurrent excitatory circuits between sprouted mossy fibers and granule cell dendrites in the inner molecular layer of the dentate gyrus (9, 28, 30). In addition, there is evidence that inhibitory nonprincipal cells also receive an input from sprouted mossy fibers (39). This study was undertaken to further characterize possible target cells for sprouted mossy fibers, using immunofluorescent staining for different calcium-binding proteins in combination with Timm histochemical staining for mossy fibers. Rats were injected intraperitoneally with kainic acid in order to induce epileptic convulsions and mossy fiber sprouting. After 2 months survival, hippocampal sections were immunostained for parvalbumin, calbindin D28k, or calretinin followed by Timm-staining. Under a fluorescent microscope, zinc-positive mossy fibers in epileptic rats were found to surround parvalbumin-containing neurons in the granule cell layer and to follow their dendrites, which extended toward the molecular layer. In addition, dendrites of calbindin D28k-containing cells were covered by multiple mossy fiber terminals in the inner molecular layer. However, the calretinin-containing cell bodies in the granule cell layer did not receive any contacts from the sprouted fibers. Electron microscopic analysis revealed that typical Timm-positive mossy fiber terminals established several asymmetrical synapses with the soma and dendrites of nonpyramidal cells within the granule cell layer. These results provide direct evidence that, in addition to recurrent excitatory connections, inhibitory circuitries, especially those responsible for the perisomatic feedback inhibition, are formed as a result of mossy fiber sprouting in experimental epilepsy.     

Extensive target cell loss during development results in mossy fibers in the regio superior (CA1) of the rat hippocampal formation

The axons of dentate granule cells (mossy fibers) have been reported to appear in the regio superior (CA1) of the rat hippocampal formation following destruction of the pyramidal cells in the regio inferior (CA3). We undertook the present experiments to confirm this finding and to determine the requirements for this dramatic neuronal rearrangement. We found that extensive (greater than 80%) loss of CA3 cells, as well as the presence of surviving CA1 neurons within a narrow period of development (postnatal days 3-5) is necessary, however apparently not sufficient, for the appearance of CA1 mossy fibers. That the absence of normal target cells during a restricted period of mossy fiber development will lead to their association with novel targets suggests that much of the specificity of this developing connection depends on the presence of normal targets during a critical period.