Jens Zimmer

Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat

The fluorescent dye 6-methoxy-8-p-toluene sulfonamide quinoline (TSQ) was used to monitor the distribution of zinc in the hippocampus and fascia dentata of adult rats subjected to 20 min of cerebral ischemia. In normal brains TSQ stains only neuropil, in particular the mossy fiber layers in the dentate hilus (CA4) and CA3, but within 2 h after ischemia, TSQ-fluorescent cells were observed in the dentate hilus. At longer survival times TSQ-positive cells stained positively with acid fuchsin, a sign of cellular degeneration. At the same time a decrease in the TSQ fluorescence of the mossy fiber terminals in the dentate hilus (CA4) and the CA3 mossy fiber layer was noted. The observations suggest that zinc many play a role in the selective death of dentate hilar neurons after cerebral ischemia.

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

SummaryModifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.

Early loss of somatostatin neurons in dentate hilus after cerebral ischemia in the rat precedes CA-1 pyramidal cell loss.

SummarySomatostatin (SS)- and cholecystokinin (CCK)-immunopositive cell somata in the rat hippocampus were quantitated at day 1, 2, 3 and 4 after cerebral ischemia. A significant (P<0.01) 60%–80% loss of hilar and CA-3c SS neurons took place. No CCK neurons were lost. Damage to SS neurons was significant on the second postischemic day and preceded the delayed loss of CA-1 neurons. We speculate that loss of SS neurons, which presumably innervate the inhibitory GABAergic (γ-aminobutyric acid) interneurons, may induce hyperactivity stimulating the Ca-1 neurons to death.

Extended commissural and ipsilateral projections in postnatally deentorhinated hippocampus and fascia dentata demonstrated in rats by silver impregnation

Abstract Within the hippocampus and the fascia dentata the different afferent systems are restricted to characteristic and often exclusive laminae. The present study describes changes in this laminar pattern following primary unilateral removal of the perforant path which conveys projections from the entorhinal area to the hippocampus and the fascia dentata. The primary operation was performed on rats 5, 9, 14, 15 and 21 days old. Sufficient time was allowed to secure removal of the debris. Following secondary lesions anterograde degeneration demonstrated by silver impregnation was used to mark the terminal fields of remaining systems. Commissural fibers passing the ventral commissure and ipsilateral fibers from CA3 of the deentorhinated hippocampus itself were found to terminate in the deafferented molecular layers of CA3 and fascia dentata. In contrast to the ipsilateral fibers the commissural ones spared a superficial brim in fascia dentata. The mutual mediolateral distribution characteristics of the normal afferents, however, tended to be retained by the extraordinary fibers. In CA1, a spread of terminals superficially in the subfield was observed, but only when the primary lesion in addition to the entorhinal area included part of the angular bundle and subiculum. Fibers passing the dorsal commissure did not extend from their normal termination in CA1 and subiculum into CA3 or fascia dentata. Of the animals operated on, those deentorhinated when 21 days old showed relative decrease in intensity of the extraordinary degeneration. The existence and role of extension of other connections into the deafferented zones are discussed. Finally the possible mechanisms underlying the aberrant growth phenomena are considered and definitions of the terms aberrant axonal growth, rerouting and sprouting and their distinctions are proposed.

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetyl cholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

Cellular, histochemical and connective organization of the hippocampus and fascia dentata transplanted to different regions of immature and adult rat brains

The aims of the present study were to examine the survival and the cellular and connective differentiation of intracerebral transplants of fascia dentata and hippocampus. Pieces of immature dentate and hippocampal tissue were taken from late embryonic (E18) and early postnatal (1-9 days old) rats and transplanted into the brains of 1- to 13-day-old and adult rats. After survival times from 4 days to 2 years the cellular and connective organization of the transplants was monitored in parallel series of sections stained with thionin (cell bodies), Timms sulphide silver method (terminal fields). Nauta and Fink-Heimer methods (normal and degenerating fibers) and a method for AChE activity (cholinergic afferents). The transplants survived well in all combinations of donor and recipient ages used, and they survived and differentiated in all parts of the recipient brains, although relations to pial and ventricular surfaces appeared to be optimal. Cell differentiation continued after transplantation, and a characteristic laminar organization was retained, although least in embryonic donor tissues. The distribution of intrinsic connections was determined by the types of subfields present in the transplants and interaction with ingrown host afferents. All aberrant intrinsic connections observed corresponded to aberrant connections formed in the hippocampus and fascia dentata denervated in situ and included supragranular mossy fibers in the fascia dentata, aberrant infrapyramidal mossy fibers in CA3, spread of CA4-associated afferents beyond the normal commissural-associational zone in the dentate molecular layer together with ingrowth of CA3-associated and CA1-subiculum-associated afferents. Most transplants received a cholinergic input of host origin irrespective of the localization in the host brain, but also non-cholinergic host pathways innervated the transplants, in particular when the transplants were in close contact with host fiber tracts, and when the recipients were immature. At various transplant locations the non-cholinergic host afferents belonged to the commissural hippocampo-dentate system, the commissural hippocampal system and the callosal system. Other cases suggested innervation of dentate transplant by host entorhinal afferents. The formation and distribution of intrinsic transplant connections and connections between transplant and host appeared to be regulated by the same factors that regulate the development and reorganization of fiber connections in the normal and the in situ denervated hippocampus and fascia dentata. As a special variety of this, the distribution of cholinergic afferents adjusted to the distribution of the major intrinsic and extrinsic non-cholinergic pathways.

Changes in the Timm sulfide silver staining pattern of the rat hippocampus and fascia dentata following early postnatal deafferentation

Abstract The Timm sulfide silver method normally stains the hippocampus and the fascia dentata of the rat in a laminar fashion, and the laminae correspond remarkably well to the terminal fields of the various afferent systems. Surgical removal of a major afferent system with origin in the entorhinal area — the perforant path — was performed in animals from 5 to 30 days old, and the effects on the Timm staining pattern analysed. The perforant path, distinguished in a lateral and a medial part, normally terminates in non-overlapping laminae in stratum moleculare (lacunosum-moleculare) of CA3 and the outer parts of the dentate molecular layer. In the deentorhinated animals these layers had only achieved between one-half and two-thirds of their normal width. Also, the staining characteristics of the perforant path zones were absent. Depending on the age of the animals at the time of the deentorhination other staining types resembling those of the unaltered neighboring fields had replaced the normal staining in the deafferented zones, either totally or in part. According to type and predominant localization the extended staining was classified as either CA3 or CA1-subiculum associated. A special staining type resembling that of the intense black mossy fibers of the hilus of fascia dentata was regularly found above the granule cell bodies in the deentorhinated animals. The replacement of the normal staining of the deafferented layers of fascia dentata and CA3 by staining types resembling those found in adjacent fields is discussed in relation to earlier observations of aberrant growth of axons terminating in these fields. A failure of decommissuration to cause any changes in the Timm staining pattern was also observed. The ipsilateral association systems which normally share terminal fields with the commissural fibers probably preserve the integrity of these common zones.

Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study

Neural mouse xenografts undergoing rejection in the adult recipient rat brain were characterized with regard to infiltrating host leukocytes and reactions of graft and host astro- and microglial cells. Rejection occurred within 35 days with infiltration of the grafts by in particular macrophages and T-cells as well as blood-brain barrier (BBB) leakage for IgG. In the surrounding host brain microglial cells showed increased histochemical staining for nucleoside diphosphatase (NDPase) and increased immunocytochemical expression of complement receptor type 3 (CR3), while astroglial cells displayed an increased immunoreactivity for glial fibrillary acidic protein (GFAP). Light microscopic findings of rat major histocompatibility complex (MHC) antigen class I on microglial cells, endothelial cells and leukocytes were confirmed at the ultrastructural level and extended to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglia. We suggest that host macrophages and activated host and xenograft microglial cells act in situ as immunostimulatory cells on T-helper cells, and that increased levels of donor MHC antigen class I may further enhance the killer activity exerted by host T-cytotoxic cells.

Commissural fibers terminate on non-pyramidal neurons in the guinea pig hippocampus — a combined Golgi/EM degeneration study

The combined Golgi/EM method was applied to guinea pig hippocampi with acute anterograde degeneration of the commissural afferents in order to identify possible synaptic contacts between commissural terminals and non-pyramidal neurons. Degenerating, electron-dense terminals of commissural origin were found in synaptic contact with both perikarya and dendrites of two identified non-pyramidal neurons in regio superior, namely a basket cell and a bipolar neuron in stratum oriens. The observed connection may form the morphological basis for the physiologically observed feed-forward inhibition of the pyramidal cells.

Development of the Hippocampus and Fascia Dentata: Morphological and Histochemical Aspects

Publisher Summary This chapter discusses the aspects of the development of the hippocampus and the fascia dentata in the rat. The emphasis is placed on the developmental changes that take place during the first postnatal month. To illustrate the different developmental stages, including the laminar differentiation of the afferent fiber systems, results obtained with the Timm sulfide silver method are used. This histochemical method provides an excellent means for monitoring and screening en bloc the development of the afferent systems to the hippocampal region. The chapter presents examples of the experimental approaches, which involve the hippocampus and the fascia dentata and help clarify which factors are important for the formation and laminar segregation of nervous connections. The chapter also presents the preliminary results obtained by transplantation of hippocampal and dentate tissue from neonatal rats into the central nervous system (CNS) of littermates and adult rats.