Hippocampal Mossy Fiber System Research Articles

Neonatal ketamine exposure impairs infrapyramidal bundle pruning and causes lasting increase in excitatory synaptic transmission in hippocampal CA3 neurons

Preclinical models demonstrate that nearly all anesthetics cause widespread neuroapoptosis in the developing brains of infant rodents and non-human primates. Anesthesia-induced developmental apoptosis is succeeded by prolonged neuropathology in the surviving neurons and lasting cognitive impairments, suggesting that anesthetics interfere with the normal developmental trajectory of the brain. However, little is known about effects of anesthetics on stereotyped axonal pruning, an important developmental algorithm that sculpts neural circuits for proper function. Here, we proposed that neonatal ketamine exposure may interfere with stereotyped axonal pruning of the infrapyramidal bundle (IPB) of the hippocampal mossy fiber system and that impaired pruning may be associated with alterations in the synaptic transmission of CA3 neurons. To test this hypothesis, we injected postnatal day 7 (PND7) mouse pups with ketamine or vehicle over 6 h and then studied them at different developmental stages corresponding to IPB pruning (PND20–40). Immunohistochemistry with synaptoporin (a marker of mossy fibers) revealed that in juvenile mice treated with ketamine at PND7, but not in vehicle-treated controls, positive IPB fibers extended farther into the stratum pyramidale of CA3 region. Furthermore, immunofluorescent double labeling for synaptoporin and PSD-95 strongly suggested that the unpruned IPB caused by neonatal ketamine exposure makes functional synapses. Importantly, patch-clamp electrophysiology for miniature excitatory postsynaptic currents (mEPSCs) in acute brain slices ex vivo revealed increased frequency and amplitudes of mEPSCs in hippocampal CA3 neurons in ketamine-treated groups when compared to vehicle controls. We conclude that neonatal ketamine exposure interferes with normal neural circuit development and that this interference leads to lasting increase in excitatory synaptic transmission in hippocampus.

Recurrent rewiring of the adult hippocampal mossy fiber system by a single transcriptional regulator, Id2

Circuit formation in the central nervous system has been historically studied during development, after which cell-autonomous and nonautonomous wiring factors inactivate. In principle, balanced reactivation of such factors could enable further wiring in adults, but their relative contributions may be circuit dependent and are largely unknown. Here, we investigated hippocampal mossy fiber sprouting to gain insight into wiring mechanisms in mature circuits. We found that sole ectopic expression of Id2 in granule cells is capable of driving mossy fiber sprouting in healthy adult mouse and rat. Mice with the new mossy fiber circuit solved spatial problems equally well as controls but appeared to rely on local rather than global spatial cues. Our results demonstrate reprogrammed connectivity in mature neurons by one defined factor and an assembly of a new synaptic circuit in adult brain.

Selective apoptosis induction in the hippocampal mossy fiber pathway by exposure to CT105, the C-terminal fragment of Alzheimer's amyloid precursor protein

β-amyloid protein (Aβ), a proteolytic byproduct of Alzheimer's amyloid precursor protein (APP), has been shown to play a central role in the development of Alzheimer's disease (AD). In addition, recent studies strongly suggest that other byproducts of proteolysis, such as C-terminal fragments of APP (APP-CTF), are also critically involved in the AD pathology. To explore this possibility, we investigated the histopathological changes induced by repeated low-dose intrahippocampal injection of a recombinant 105 amino acid C-terminal fragment of APP (CT105). First, we carried out a behavioral analysis by using the three-panel runway task, and found that the working memory was significantly impaired by CT105 exposure. Then, via propidium iodide staining, we encountered a number of cells exhibiting fragmented or shrank nuclei in the mossy fiber pathway (stratum lucidum and dentate hilus) in CT105-treated rats. These cells were positive for single-stranded DNA (ssDNA), an apoptosis-specific marker, and thus were considered to be apoptotic. Some of the ssDNA-positive cells were also positive for somatostatin. But neither ionized calcium-binding adapter molecule 1 (Iba1) nor S100β occurred in ssDNA-positive cells. These findings suggest that CT105 induces apoptotic changes in cells of neuronal origin. Quantitative analysis showed that the densities of ssDNA-positive cells in the mossy fiber pathway were significantly higher in CT105-treated rats than in control animals. The present results suggest that CT105 causes dysfunction in the hippocampal mossy fiber system, and also provide some key to understand the relationship between APP-CTF and glutamatergic synaptic dysregulation in AD.

Spatial learning induces presynaptic structural remodeling in the hippocampal mossy fiber system of two rat strains

Hebb (1949) proposed that after learning both presynaptic and postsynaptic structural changes form the neural substrate of long-lasting memory. Despite this, there are few instances linking presynaptic remodeling with learning. Here the authors demonstrate in two different rat strains that learning the location of a hidden platform induces expansion of the presynaptic hippocampal mossy fiber terminal field (MFTF) from the stratum lucidum to the distal stratum oriens (dSO). Prior to any training, Long Evans rats (LER) showed an extensive endogenous MFTF innervation of DSO, in contrast to Wistar rats (WR) that showed minimal innervation. LER showed better recall than WR on the hidden platform water maze task and a visible reversal water maze task. In both strains, significant MFTF expansion to dSO, spanning approximately 200 mum, was detected 7 days after training on the hidden platform task, but only LER showed significant MFTF expansion 24 h after training. It is attractive to think that the MFTF expansion to dSO contributes both to long-lasting memory formation and to facilitating spatial navigation strategies. The present results establish learning-induced axonal remodeling of the hippocampal MF system in adult rats as an especially useful system for exploring presynaptic morphological adjustments consequent to learning.

Activity-dependent changes in synaptophysin immunoreactivity in hippocampus, piriform cortex, and entorhinal cortex of the rat

Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy fiber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy fiber cortical pathways in the adult rat. We found a significant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no significant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that different populations of sprouting axons are labeled by these two techniques, and suggests that activation-induced sprouting extends well beyond the hippocampal mossy fiber system.

Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications.

The effects of EGb 761 on the CNS underlie one of its major therapeutic indications; i.e., individuals suffering from deteriorating cerebral mechanisms related to age-associated impairments of memory, attention and other cognitive functions. EGb 761 is currently used as symptomatic treatment for cerebral insufficiency that occurs during normal ageing or which may be due to degenerative dementia, vascular dementia or mixed forms of both, and for neurosensory disturbances. Depressive symptoms of patients with Alzheimer's disease (AD) and aged non-Alzheimer patients may also respond to treatment with EGb 761 since this extract has an "anti-stress" effect. Basic and clinical studies, conducted both in vitro and in vivo, support these beneficial neuroprotective effects of EGb 761. EGb 761 has several major actions; it enhances cognition, improves blood rheology and tissue metabolism, and opposes the detrimental effects of ischaemia. Several mechanisms of action are useful in explaining how EGb 761 benefits patients with AD and other age-related, neurodegenerative disorders. In animals, EGb 761 possesses antioxidant and free radical-scavenging activities, it reverses age-related losses in brain alpha 1-adrenergic, 5-HT1A and muscarinic receptors, protects against ischaemic neuronal death, preserves the function of the hippocampal mossy fiber system, increases hippocampal high-affinity choline uptake, inhibits the down-regulation of hippocampal glucocorticoid receptors, enhances neuronal plasticity, and counteracts the cognitive deficits that follow stress or traumatic brain injury. Identified chemical constituents of EGb 761 have been associated with certain actions. Both flavonoid and ginkgolide constituents are involved in the free radical-scavenging and antioxidant effects of EGb 761 which decrease tissue levels of reactive oxygen species (ROS) and inhibit membrane lipid peroxidation. Regarding EGb 761-induced regulation of cerebral glucose utilization, bilobalide increases the respiratory control ratio of mitochondria by protecting against uncoupling of oxidative phosphorylation, thereby increasing ATP levels, a result that is supported by the finding that bilobalide increases the expression of the mitochondrial DNA-encoded COX III subunit of cytochrome oxidase. With regard to its "anti-stress" effect, EGb 761 acts via its ginkgolide constituents to decrease the expression of the peripheral benzodiazepine receptor (PBR) of the adrenal cortex.

PSA-NCAM: an important regulator of hippocampal plasticity

The Neural Cell Adhesion Molecule (NCAM) serves as a temporally and spatially regulated modulator of a variety of cell-cell interactions. This review summarizes recent results of studies aimed at understanding its regulation of expression and biological function, thereby focussing on its polysialylated isoforms (PSA-NCAM). The detailed analysis of the expression of PSA and NCAM in the hippocampal mossy fiber system and the morphological consequences of PSA-NCAM deficiency in mice support the notion that the levels of expression of NCAM are important not only for the regulation and maintenance of structural changes, such as migration, axonal growth and fasciculation, but also for activity-induced plasticity. There is evidence that PSA-NCAM can specifically contribute to a presynaptic form of plasticity, namely long-term potentiation at hippocampal mossy fiber synapses. This is consistent with previous observations that NCAM-deficient mice show deficits in spatial learning and exploratory behavior. Furthermore, our data points to an important role of the hypothalamic–pituitary–adrenal axis, which is the principle adaptive response of the organism to environmental challenges, in the control of PSA-NCAM expression in the hippocampal formation. In particular, we evidence an inhibitory influence of corticosterone on PSA-NCAM expression.

Modulation of mRNA expression of the neurotrophins of the nerve-growth-factor family and their receptors in the septum and hippocampus of rats after transient postnatal thyroxine treatment. II. Effects on p75 and trk receptor expression.

Early postnatal application of thyroid hormones to rats results in morphological changes of the septo-hippocampal cholinergic and the hippocampal mossy fiber systems. Modulation in the expression of either neurotrophins and/or their receptors is postulated to be involved in these effects. In a recent study, we showed that, after thyroxine application, the mRNA expression of neurotrophins of the nerve-growth-factor (NGF) family is significantly upregulated both in septum and hippocampus. To test whether the neurotrophin receptors (the low-affinity neurotrophin receptor p75 and the specific high-affinity receptors trkA, trkB, and trkC) were also affected by hormone administration, newborn rats were treated daily with subcutaneous injections of thyroxine until postnatal day 12 (P12) at latest. Control animals received corresponding injections of saline. The pups were sacrificed at defined intervals from P9 to P14. The septal areas and the hippocampi were analyzed using the reverse-transcription polymerase chain reaction (RT-PCR) method for quantification of p75, trkA, trkB, and trkC mRNA levels. Analysis of variance over the total investigation period revealed no significant general increases of the gene expressions of either neurotrophin receptor, neither in the septum nor in the hippocampus, although previous results have shown marked changes in neurotrophin levels. On particular postnatal days, significant upregulation could be observed in hippocampus for trkB and trkC. From these and recent data, we conclude that modulation of neurotrophin expression rather than neurotrophin-receptor expression contributes to the morphological modifications within the hippocampal mossy fiber system and the septo-hippocampal cholinergic system.

Long-term but not short-term plasticity at mossy fiber synapses is impaired in neural cell adhesion molecule-deficient mice.

Cell adhesion molecules (CAMs) are known to be involved in a variety of developmental processes that play key roles in the establishment of synaptic connectivity during embryonic development, but recent evidence implicates the same molecules in synaptic plasticity of the adult. In the present study, we have used neural CAM (NCAM)-deficient mice, which have learning and behavioral deficits, to evaluate NCAM function in the hippocampal mossy fiber system. Morphological studies demonstrated that fasciculation and laminar growth of mossy fibers were strongly affected, leading to innervation of CA3 pyramidal cells at ectopic sites, whereas individual mossy fiber boutons appeared normal. Electrophysiological recordings performed in hippocampal slice preparations revealed that both basal synaptic transmission and two forms of short-term plasticity, i.e., paired-pulse facilitation and frequency facilitation, were normal in mice lacking all forms of NCAM. However, long-term potentiation of glutamatergic excitatory synapses after brief trains of repetitive stimulation was abolished. Taken together, these results strongly suggest that in the hippocampal mossy fiber system, NCAM is essential both for correct axonal growth and synaptogenesis and for long-term changes in synaptic strength.

Biosynthesis and metabolism of native and oxidized neuropeptide Y in the hippocampal mossy fiber system.

Neuropeptide Y (NPY) gene expression is known to be modulated in the mossy fiber projection of hippocampal granule cells following seizure. We investigated NPY biosynthesis and metabolism in an attempt to characterize NPY biochemically as a neurotransmitter in the granule cell mossy fiber projection. NPY biosynthesis was compared in normal control animals and in animals that had experienced a single pentylenetetrazole-induced seizure. In situ hybridization analysis established the postseizure time course of preproNPY mRNA expression in the hippocampal formation, localizing the majority of increased preproNPY mRNA content to the hilus of the dentate gyrus. Radioimmunoassay analysis of the CA3/mossy fiber terminal subfield confirmed a subsequent increase in NPY peptide content. Biosynthesis of NPY peptide by granule cells and transport to the CA3/mossy fiber subfield was demonstrated by in vivo radiolabel infusion to the dentate gyrus/hilus followed by sequential HPLC purification of identified radiolabeled peptide from the CA3/mossy fiber terminal subfield. Additional in vivo radiolabeling studies revealed a postseizure increase in an unidentified NPY-like immunoreactive (NPY-LI) species. HPLC/radioimmunoassay analyses of CA3 subfield tissue extracts comparing normal control animals and pentylenetetrazole-treated animals confirmed the increased total NPY-LI, and demonstrated that the increased NPY-LI was comprised of a minor increase in native NPY and a major increase in the unknown NPY-LI. Data from subsequent and separate analyses incorporating immunoprecipitation with anti-C-terminal flanking peptide of NPY, further HPLC purification, and matrix-assisted laser desorption/ionization mass spectrometry support the conclusion that the unknown NPY-LI is methionine sulfoxide NPY. NPY and NPY-sulfoxide displayed differential calcium sensitivity for release from mossy fiber synaptosomes. Similar to NPY, NPY sulfoxide displayed high-affinity binding to each of the cloned Y1, Y2, Y4, and Y5 receptor subtypes. Postrelease inactivation of NPY was demonstrated in a mossy fiber synaptosomal preparation. Thus, the present study in combination with previously reported electrophysiological activity of NPY in the CA3 subfield demonstrates that NPY fulfills the classical criteria for a neurotransmitter in the hippocampal granule cell mossy fiber projection, and reveals the presence of two molecular forms of NPY that display differential mechanisms of release while maintaining similar receptor potencies.

Modulation of mRNA expression of the neurotrophins of the nerve growth factor family and their receptors in the septum and hippocampus of rats after transient postnatal thyroxine treatment. I. Expression of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin 4 mRNA.

Early postnatal application of thyroid hormones to rats results in morphological changes in septum and hippocampus. Modulation in the expression of either neurotrophins and/or their receptors is postulated to be responsible for these effects. In the present study we tested whether thyroxine administration leads to changes in the expression of neurotrophins of the nerve growth factor (NGF) family. Newborn rats were treated daily with subcutaneous injections of thyroxine until postnatal day (P) 12 at maximum. The pups were killed at defined intervals from P2 to 21. The septal area and the hippocampi were analyzed using the reverse transcriptase-PCR method for quantitation of NGF, brain-derived neurotrophic factor (BDNF), NT-3, and NT-4 messenger RNA (mRNA) levels. In hippocampus of hyperthyroid rats, as compared to controls, we found higher levels of BDNF and NT-3 mRNA over the total investigation period, whereas in the septum a thyroxine-dependent increase in NT-3 mRNA expression was observed. In addition, significant thyroxine-induced effects were found for all variables (except for NGF in the septum) at particular postnatal days. From these data we conclude that modulation of neurotrophin expression is a possible mechanism for the morphological modifications within the hippocampal mossy fiber system and the septohippocampal cholinergic system.

Hippocampal mossy fiber system in genetically epileptic rats studied using immunohistochemistry and neurotracing techniques

Hippocampal mossy fiber system in genetically epileptic rats studied using immunohistochemistry and neurotracing techniques

911 Hippocampal mossy fiber system in genetically epileptic rats studied using immunohistochemistry and neurotracing techniques

Individual differences in novelty-induced exploratory activity identify rats which can serve as a model of human sensation-seeking, risk-taking behaviour. Experimentally naı̈ve rats, when exposed to mild stress of a novel environment, exhibit variability in their exploratory activity. Some rats display high rates of locomotor reactivity to novelty (high responders (HR)), and others display low rates (low responders (LR)). The LRHR phenotype is a reliable predictor of drug-taking behaviour and is linked to differences in hippocampal glucocorticoid receptor mRNA expression. In this study, we investigated whether the LRHR phenotype is associated with differences in the quantitative morphology of the hippocampal field CA3, dentate gyrus molecular layer, granule cell layer and mossy fibres. LRs and HRs showed no significant differences in the volumes of CA3 and dentate molecular layer volume or the number of dentate granule cells. However, LRs had a significantly larger suprapyramidal mossy fibre terminal field volume when compared to HRs. The infrapyramidal mossy fibres did not differ between phenotypes. Also, we found a LRHR phenotype-independent significant negative correlation between molecular layer volume per granule cell and the total number of granule cells. These findings implicate the SP-MF in vulnerability for risk-taking behaviour, and we propose that LR and HR hippocampi may differ in the way novelty information is processed.

Neuroethological and morphological (Neo-Timm staining) correlates of limbic recruitment during the development of audiogenic kindling in seizure susceptible Wistar rats.

Acute audiogenic seizures are a model of generalized tonic-clonic seizures, induced by high intensity acoustic stimulation in genetically susceptible rodents. The neural substrate are sensory motor brainstem nuclei. Recruitment of forebrain structures takes places upon repetition of acoustically evoked seizures. The term audiogenic kindling means forebrain kindling evoked by repeated brainstem seizures and has been described in several strains of genetically epilepsy-prone rats. Thus, the present work was conducted in order to test the hypothesis that audiogenic kindling recruits the forebrain, which may be behaviorally evaluated and associated with morphological changes as well. The behavioral sequences observed during the development of audiogenic kindling were assessed by neuroethological methods (cluster analysis), with the ETHOMATIC program. Seizure severity indexes (brainstem and limbic seizures) and latencies of wild running and tonic-clonic seizures were measured to quantify seizure evolution. Densitometric analysis of Neo-Timm staining was used for assessing morphological changes associated with audiogenic kindling. In group I, II resistant (R) and 16 susceptible (S) animals were stimulated (120 dB) 21 times, and allowed a 10 day recovery period prior to retesting. In group II, 22 R and 20 S were stimulated 60 times, and allowed a 2 month recovery period prior to retesting. Repetition of the acoustic stimulation in group I and group II susceptible animals led to a progressive and statistically significant attenuation of the behaviors associated with brainstem seizures and a concomitant increased expression of the behaviors associated with limbic seizures. After either a 10 day (group I) or 2 month (group II) recovery period, acoustic stimulation preferentially evoked brainstem-associated behaviors and seizures rather than limbic ones in the audiogenic susceptible animals, although in some animals overlapped brainstem and limbic seizures were detected. Latencies for the wild running and tonic seizures after acoustic stimulation significantly increased during audiogenic kindling for both group I and group II susceptible animals. The quantitative ethological evaluation in both group I and group II, illustrated by flowcharts, showed the evolution of the kindling installation by the presence of limbic seizure clusters, competing in time with the original tonic-clonic clusters. Expression of limbic seizures by group I animals, after acoustic stimulation, was not associated with changes in the mossy fiber Neo-Timm staining pattern of these animals. In group II however, Neo-Timm staining revealed mossy fiber sprouting in the ventral hippocampus (but not in the dorsal), and a significant change in the optical density of amygdaloid nuclei and perirhinal cortex in susceptible animals as compared to resistant ones. In conclusion, audiogenic kindling effectively recruits forebrain structures, responsible for the appearance of limbic seizures. It is possible that the paradigm used in group I was subthreshold for the development of clear-cut synaptic reorganization in the hippocampal mossy fiber system, since the behavioral patterns reverted ten days after the last seizure induction. In group II, however, an increased number of evoked seizures and a more prolonged time after the last chronic seizure showed structural re-arrangements in amygdala, perirhinal cortex and hippocampus, associated with permanence in terms of behavioral data (lack of regression of limbic seizures to control values).

Altered patterns of dynorphin immunoreactivity suggest mossy fiber reorganization in human hippocampal epilepsy

Dynorphin A(1-17), an opioid peptide that is normally present in the hippocampal mossy fiber system, was localized immunocytochemically in the hippocampal formation of control autopsy and temporal lobe epilepsy (TLE) specimens. In control tissue, dynorphin-like immunoreactive (Dyn-IR) structures were confined to the mossy fiber path and were most highly concentrated in the polymorph (hilar) region of the dentate gyrus. Very few Dyn-IR structures were present in the molecular and granule cell layers of the dentate gyrus. In contrast, in all TLE specimens, Dyn-IR elements were present in these layers. The extent of aberrant staining varied among the TLE specimens, and 2 major patterns were observed. The first was a relatively wide band of reaction product in the inner one-third to one-fourth of the molecular layer (8 cases), and the second was a more limited distribution of immunoreactive fibers and presumptive terminals in the granule cell and immediately adjacent supragranular regions (2 cases). The extent of aberrant Dyn-IR structures appeared to be related to the amount of cell loss in the polymorph and CA3 fields and to dispersion of the granule cell somata. Specimens processed with the Timm's sulfide silver method for heavy metals provided independent evidence for the distribution of mossy fibers. In both control and TLE specimens, the patterns of labeling were virtually identical to those of dynorphin localization. These findings suggest that sprouting of mossy fibers or their axon collaterals has occurred in hippocampal epilepsy and that the reorganized fibers contain at least one of the neuropeptides that are normally present in this system. Such fibers could form recurrent excitatory circuits and contribute to synchronous firing and epileptiform activity, as suggested in studies of experimental models of epilepsy.

Seizures induce dramatic and distinctly different changes in enkephalin, dynorphin, and CCK immunoreactivities in mouse hippocampal mossy fibers

Light microscopic immunocytochemical techniques were used to evaluate the influence of recurrent limbic seizure activity on the immunoreactivity for 3 neuropeptides--enkephalin, dynorphin, and cholecystokinin (CCK)--contained within the mouse hippocampal mossy fiber axonal system. Seizures were induced either by the placement of a small unilateral electrolytic lesion in the dentate gyrus hilus or by intraventricular injection of kainic acid. Both treatments induce epileptiform activity in hippocampus lasting several hours. Four days after either lesion placement or injection of 0.05-0.1 microgram kainic acid, immunoreactivity for all 3 peptides was altered throughout the intact mossy fiber system, bilaterally, but in distinctly different ways: enkephalin immunoreactivity (ENK-I) was dramatically elevated, dynorphin immunoreactivity was reduced, and CCK immunoreactivity (CCK-I) was either severely reduced or completely absent in the mossy fiber system. ENK-I was also clearly increased in other areas, including the lateral septum, the entorhinal cortex, and within the entorhinal (perforant path) efferents to temporal hippocampus. In contrast, the loss of CCK seemed restricted to the mossy fiber system in that immunostaining appeared normal in scattered hippocampal perikarya, within the dentate gyrus commissural system, as well as within other limbic structures. Four days after injections of 0.2 or 0.25 microgram kainic acid, mossy fiber ENK-I was greatly elevated, dynorphin immunoreactivity was reduced, but, unlike the situation with lower kainic acid doses, CCK-I was only modestly reduced in the mossy fibers and was clearly reduced in other hippocampal systems as well. These data indicate that epileptiform physiological activity differentially affects the regulation of 3 neuroactive peptides contained within the hippocampal mossy fiber system and suggest a mechanism through which seizurelike episodes can have a lasting influence on the operation of specific hippocampal circuitries.

Focal hippocampal lesions induce seizures and long-lasting changes in mossy fiber enkephalin and CCK immunoreactivity

Electrolytic lesions of the dentate gyrus hilus have been demonstrated to induce behavioral seizure activity and to result in perturbations in the amount of enkephalin, cholecystokinin, and dynorphin immunoreactivity in the hippocampal mossy fiber system. In the present study, electroencephalographic (EEG) recordings, made from hippocampus contralateral to a hilus lesion in mouse, demonstrate the presence of recurrent hippocampal seizure activity which begins approximately one hour postlesion and continues for several hours thereafter. Behavioral seizures were found to correspond to periods of epileptiform hippocampal EEG. Immunocytochemical analyses of enkephalin-(ENK-I) and cholecystokinin-immunoreactivity (CCK-I) in contralateral hippocampus of animals sacrificed at various postlesion intervals revealed that both ENK-I and CCK-I were depleted from the mossy fibers at 6 and 12 hr postlesion, and that ENK-I rebounded to supranormal levels by 27 hr. In two animals sacrificed 60 days following lesions which induced extreme behavioral seizure activity, ENK-I was still elevated while CCK-I was completely absent from the mossy fiber system. These data suggest that heightened physiological activity, in the form of recurrent limbic seizures, induces long-lasting but quite different alterations in enkephalin and CCK concentration in the hippocampal mossy fiber system.

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.

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.

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 region 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 micrometer above to approximately 1,600 micrometer 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.