Thomas Naumann

Hippocampal Synapses Depend on Hippocampal Estrogen Synthesis

Estrogens have been described to induce synaptogenesis in principal neurons of the hippocampus and have been shown to be synthesized and released by exactly these neurons. Here, we have focused on the significance of local estrogen synthesis on spine synapse formation and the synthesis of synaptic proteins. To this end, we reduced hippocampal estrogen synthesis in vitro with letrozole, a reversible nonsteroidal aromatase inhibitor. In hippocampal slice cultures, letrozole treatment resulted in a dose-dependent decrease of 17β-estradiol as quantified by RIA. This was accompanied by a significant decrease in the density of spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a downregulation of spinophilin, a marker of dendritic spines, and synaptophysin, a protein of presynaptic vesicles, in response to letrozole. Surprisingly, no increase in the density of spines, boutons, and synapses and in spinophilin expression was seen after application of estradiol to the medium of cultures that had not been treated with letrozole. However, synaptophysin expression was upregulated under these conditions. Our results point to an essential role of endogenous hippocampal estrogen synthesis in the maintenance of hippocampal spine synapses.

Neuronal 3′,3,5-Triiodothyronine (T3) Uptake and Behavioral Phenotype of Mice Deficient in Mct8, the Neuronal T3 Transporter Mutated in Allan–Herndon–Dudley Syndrome

Thyroid hormone transport into cells requires plasma membrane transport proteins. Mutations in one of these, monocarboxylate transporter 8 (MCT8), have been identified as underlying cause for the Allan–Herndon–Dudley syndrome, an X-linked mental retardation in which the patients also present with abnormally high 3′,3,5-triiodothyronine (T3) plasma levels. Mice deficient in Mct8 replicate the thyroid hormone abnormalities observed in the human condition. However, no neurological deficits have been described in mice lacking Mct8. Therefore, we subjected Mct8-deficient mice to a comprehensive immunohistochemical, neurological, and behavioral screen. Several behavioral abnormalities were found in the mutants. Interestingly, some of these behavioral changes are compatible with hypothyroidism, whereas others rather indicate hyperthyroidism. We thus hypothesized that neurons exclusively dependent on Mct8 are in a hypothyroid state, whereas neurons expressing other T3 transporters become hyperthyroid, if they are exposed directly to the high plasma T3. The majority of T3 uptake in primary cortical neurons is mediated by Mct8, but pharmacological inhibition suggested functional expression of additional T3 transporter classes. mRNAs encoding six T3 transporters, including L-type amino acid transporters (LATs), were coexpressed with Mct8 in isolated neurons. We then demonstrated Lat2 expression in cultured neurons and throughout murine brain development. In contrast, LAT2 is expressed in microglia in the developing human brain during gestation, but not in neurons. We suggest that lack of functional complementation by alternative thyroid hormone transporters in developing human neurons precipitates the devastating neurodevelopmental phenotype in MCT8-deficient patients, whereas Mct8-deficient mouse neurons are functionally complemented by other transporters, for possibly Lat2.

Up-regulation of astrocyte-derived tenascin-C correlates with neurite outgrowth in the rat dentate gyrus after unilateral entorhinal cortex lesion

The extracellular matrix protein tenascin-C has been implicated in the regulation of axonal growth. Using unilateral entorhinal cortex lesions, which induce a massive sprouting response in the denervated outer molecular layer of the rat fascia dentata, the role of tenascin-C for axonal growth was investigated in vivo. Monoclonal antibodies against the neurite outgrowth and anti-adhesive domains of the molecule were employed. Immunostaining was increased throughout the denervated outer molecular layer by day 2, reached a maximum around day 10, and was back to control levels by four weeks post lesion. Growth cone deflecting as well as neurite outgrowth promoting isoforms of tenascin-C were up-regulated after the lesion. Using electron microscopy, single intensely tenascin-C immunoreactive cells were identified as reactive astrocytes that phagocytose degenerated terminals. In situ hybridization histochemistry for tenascin-C messenger RNA revealed numerous cellular profiles in the denervated outer molecular layer of the ipsilateral and contralateral dentate gyrus two days post lesion. Tenascin-C messenger RNA-positive cells in the outer molecular layer were identified as astrocytes using double-labelling for tenascin-C messenger RNA and glial fibrillary acidic protein immunohistochemistry. Thus, a tenascin-C-rich substrate is present in the outer molecular layer during the time of sprouting and a sharp boundary is formed against the inner molecular layer. This pattern may contribute to the layer-specific sprouting response of surviving afferents after entorhinal lesion. Neurite outgrowth may be promoted within the denervated zone, whereas axons trying to grow into the denervated outer molecular layer, for example from the inner molecular layer, would be deflected by a tenascin-C-rich barrier.

Retrograde tracing with Fluoro-Gold: different methods of tracer detection at the ultrastructural level and neurodegenerative changes of back-filled neurons in long-term studies

Among the available retrograde fluorescent tracers Fluoro-Gold (FG) is particularly advantageous because it (1) is not only detectable by fluorescence microscopy but also immunocytochemically, resulting in an almost complete staining of the dendritic arbor, (2) is visible in lysosome-like structures allowing for the identification of projection neurons at the ultrastructural level, and (3) remains in the labeled neurons for extended periods of time. Photoconversion and immunostaining for FG, respectively, result in a stable, electron-dense reaction product. Thus, the retrogradely labeled cells can be analyzed quantitatively in the light- and electron microscope for their structural characteristics and input synapses. Long-term studies of back-filled neurons provided evidence for neurotoxic effects of FG in these cells.

Selective in vivo fluorescence labelling of cholinergic neurons containing p75NTR in the rat basal forebrain

The cholinergic system of the rat basal forebrain is used as a model for the homologous region in humans which is highly susceptible to neuropathological alterations as in Alzheimers disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75NTR. This has been utilized for selective immunolesioning of cholinergic neurons after internalization of an immunotoxin composed of anti-p75NTR and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in the basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75NTR with the bright red fluorochrome carbocyanine 3 (Cy3). Three days after intraventricular injection of Cy3-192IgG the fluorescence microscopic analysis revealed a pattern of Cy3-labelled cells matching the distribution of cholinergic neurons. Apparently the marker was internalized within complexes of p75NTR and Cy3-192IgG which were then retrogradely transported to the cholinergic perikarya of the basal forebrain. In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. The specificity of the in vivo staining was proven by subsequent immunolabelling of choline acetyltransferase (ChAT) with green fluorescent Cy2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-192IgG. In parallel experiments, digoxigenylated 192IgG was not detectable within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalized. On the other hand, digoxigenylated 192IgG was found to be an excellent immunocytochemical marker for p75NTR as shown by double labelling including highly sensitive mouse antibodies directed against ChAT. Based on the present findings, future applications of the apparently non-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed.

Identified septohippocampal neurons survive axotomy: a fine-structural analysis in the rat.

Previous studies have indicated that interruption of the connections between the medial septum and hippocampus by cutting the axons results in degeneration and death of the projecting septal neurons. However, in these studies cell death has been inferred primarily from the loss of immunoreactivity for transmitter-specific enzymes. In the present study, we labeled septohippocampal projection neurons by retrograde tracing and then cut their axons. Subsequent intracellular injection of prelabeled cells revealed the morphology of the soma and dendrites and allowed us to examine the ultrastructure of these neurons. A large number of septohippocampal neurons survived even 10 weeks after axotomy, suggesting that axotomized septohippocampal neurons survive for considerable periods beyond the time at which they stop expressing transmitter-specific immunoreactivity. Survival of axotomized neurons is a prerequisite for pharmacological interference aimed at reactivating transmitter expression, axonal re-growth, and the eventual reintegration into functionally relevant circuitries.

Development of cholinergic and GABAergic neurons in the rat medial septum: Different onset of choline acetyltransferase and glutamate decarboxylase mRNA expression

In the present study, we have investigated the developmental expression of the transmitter‐synthesizing enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in rat medial septal neurons by using in situ hybridization histochemistry. In addition, we have employed immunostaining for ChAT and the calcium‐binding protein parvalbumin, known to be contained in septohippocampal GABAergic neurons.

Is there a long-lasting effect of a short-term nerve growth factor application on axotomized rat septohippocampal neurons ?

Loss of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum (MS) following fimbria transection can be prevented by nerve growth factor (NGF) application. Here we have studied the long-term effects of a short-term NGF treatment starting immediately after lesion and lasting for the first 3 weeks. We demonstrate that this NGF treatment rescues many ChAT neurons after short survival time (3 weeks) but does not have a long-lasting (6 months) effect on both ChAT- and parvalbumin-immunopositive (GABAergic) MS neurons.

Region-specific activation of microglial cells in the rat septal complex following fimbria-fornix transection

Studies of postlesional microglial activation may gain insight into microglia/neuronal interactions in processes of neurodegeneration. We compared the microglial response after axotomy of septohippocampal projection neurons with that seen after selective immunolesioning of cholinergic septohippocampal neurons with the immunotoxin 192 IgG‐saporin. Using the microglial marker isolectin B4 from Griffonia simplicifolia (GSA I‐B4), we found striking differences in the microglial response between these two lesion paradigms. Following axotomy of septohippocampal neurons by fimbria‐fornix transection (ff‐t), there was only a moderate and short‐lasting microglial reaction in the medial septum (MS) in the early postlesion period. Prelabeling of septohippocampal neurons with Fluoro‐Gold (FG) prior to axotomy revealed the survival of most neurons, and only very rarely were microglial cells observed that had phagocytosed FG‐labeled debris. In the lateral septum (LS) containing the degenerating terminals of hippocamposeptal fibers transected by ff‐t, a heavy reaction of lectin‐labeled activated microglial cells associated with high phagocytotic activity was noticed. Unexpectedly, after a long survival time (6 months) following ff‐t, we observed an increase in microglial GSA I‐B4 labeling in the MS. In contrast, an inverse pattern of the microglial response, i.e., a strong initial reaction in the MS and very little microglial activation in the LS, was observed after immunolesioning. Our results indicate that the microglial reaction in the MS following ff‐t differs substantially from that seen in other models of axotomy. J. Comp. Neurol. 390:481–496, 1998.

Transient Up‐regulation of Ciliary Neurotrophic Factor Receptor‐α mRNA in Axotomized Rat Septa1 Neurons

Using non‐radioactive in situ hybridization we investigated the effect of fimbria‐fornix transection on the expression of ciliary neurotrophic factor receptor α (CNTFRα) mRNA in axotomized septohippocampal neurons of the rat septal complex. Whereas CNTFRα expression was undetectable in the medial septal nucleus/diagonal band complex (MSDB) of control animals, specific up‐regulation was observed in MSDB neurons after fimbria‐fornix transection. CNTFRα expression was maximal 7–10 days after the lesion and had returned to control levels after 3 weeks. Following unilateral fimbria‐fornix transection, CNTFRα up‐regulation was restricted to the MSDB ipsilateral to the lesion. When cholinergic septal neurons were selectively eliminated by immunolesioning with 192 IgG‐saporin prior to fimbria‐fornix transection, the lesion‐induced expression of CNTFRα was still observed in many medial septal nucleus neurons. These results demonstrate that after fimbria‐fornix transection CNTFRα expression is transiently induced in axotomized, non‐cholinergic neurons of the medial septal nucleus, suggesting a postlesion function of locally supplied CNTF.