gms | German Medical Science

23. Wissenschaftliche Jahrestagung der Deutschen Gesellschaft für Phoniatrie und Pädaudiologie e. V.

Deutsche Gesellschaft für Phoniatrie und Pädaudiologie e. V.

15. - 17.09.2006, Heidelberg

Goldschmidt staining of auditory cortical fields - the new method of Thallium autometallography applied around onset of hearing in juvenile Mongolian gerbils (Meriones unguiculatus)

Poster

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  • corresponding author presenting/speaker Hergen Friedrich - Sensory Development and Voice Research Group, Department of Phoniatrics and Pedaudiology, Georg-August-University Göttingen, Göttingen, Germany
  • Jürgen Goldschmidt - Auditory Learning and Speech Research Group, IfN Leibniz Institute for Neurobiology, Magdeburg, Germany
  • Ingo W. Stuermer - Sensory Development and Voice Research Group, Department of Phoniatrics and Pedaudiology, Georg-August-University Göttingen, Göttingen, Germany

Deutsche Gesellschaft für Phoniatrie und Pädaudiologie. 23. Wissenschaftliche Jahrestagung der Deutschen Gesellschaft für Phoniatrie und Pädaudiologie. Heidelberg, 15.-17.09.2006. Düsseldorf, Köln: German Medical Science; 2006. Doc06dgppP12

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Veröffentlicht: 5. September 2006

© 2006 Friedrich et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Abstract

Investigations on the development of functional activity in the auditory cortex (AC) of non-human mammals give insights into the principles of map formation. The plasticity of cortical maps suffered from auditory deprivation was already described with Fluoro-2-deoxyglucose (FDG) in domesticated Gerbils (Meriones unguiculatus forma domestica [1]). A new method to map functional activity, named Thallium (TL) autometallography, was recently developed by Goldschmidt [2]. Here, we apply this technique in juvenile gerbils to visualize map formation around hearing onset.

TL autometallography is based on the tight coupling of neuronal activity and potassium K+ uptake. Neurons have to compensate for the loss of potassium, TL as its analogue accumulates in active cells. TL distribution in cortical layers could be mapped with staining based on silver intensification of heavy metal sulphides [3]. Onset of hearing in gerbils takes place around postnatal day 14. Specimen between P11 and P17 were injected with 50-200 µl of the K+ analogue TL i.p. and kept without further sensory stimulation. Brains were removed, frozen and sectioned (25 µm). Neuronal activity marked by TL was compared with activity patterns obtained by the FDG method.

TL applied to juveniles gave sufficient visualisation of cortical activity, including the spatial resolution of cortical columns. Prominent activity in the primary auditory field (A1) and the anterior auditory field (AAF) were evident in TL as well as in FDG images. Methodological restrictions arose neither from small hearts perfused (<100 mg) nor from histology in premature brains. Ongoing research should allow the visualization of neuronal activity with cellular resolution.


Text

Introduction

Investigations on the development of functional activity in the auditory cortex of non-human mammals give insights into the principles of cortical map formation. The plasticity of tonotopic maps suffered from auditory deprivation was described in rodents with immunohistological methods and the 14-C-Fluoro-2-Desoxyglucose (FDG) technique [1]. Mongolian Gerbils (Meriones unguiculatus) and domesticated Laboratory Gerbils (Meriones unguiculatus forma domestica) proofed to be suitable animals models to study functional development of hearing. A new method for mapping neuronal activity in the mammalian brain, named thallium autometallography, was recently developed and introduced by Goldschmidt [2]. Here, we apply this technique in juvenile Mongolian gerbils to visualize map formation around hearing onset.

Material and Methods

Thallium autometallography is based on the tight coupling of neuronal activity and potassium K+ uptake within neurons. The efflux of K+ increases upon depolarisation. Neurons have to compensate for the loss of potassium by a likewise increased influx, or reuptake respectively, which is mediated to a large degree by the Na+-K+-ATPase. Due to this physiology thallium as a potassium analogue seems to cumulate in the active regions of the cortex. The distribution of thallium in different layers of the neocortex could be mapped with the help of a modified Timm-staining due to silver intensification of heavy metal sulphides that form when the blood vessels are perfused with sulphide solution [3]. In addition the method was also suitable to display endogeneous zinc pools in the brains of mammals.

Onset of hearing in Mongolian gerbils takes place at the end of the second postnatal week. Subadult specimen between postnatal day 11 (P 11) and P 17 were injected with 0.05-0.20 ml of the K+ analogue thallium i.p. and kept in a standard laboratory environment for 5 min without further acoustic or visual stimulation. Brains were removed within 9 min after perfusion, cryoptoprotected in 30% sucrose and frozen in liquid 2-methylbutan. Sections (each 0.025 mm thick) were done on a cryostat and mounted on gelatine coated glass slides. Brain sections were treated with HCl for 30 min. Zinc sulfide ZnS can easily be removed from sliced brains via a significant reduction of the pH value. Therefore staining becomes specific for Thallium sulfide (Tl2S), which forms an insoluble acid resistant precipitate in an acid environment.

Sections were digitized and analyses with customized imaging software (NIH image). Neuronal activity marked by thallium was compared with activity patterns obtained by the FDG method. Control slides were received from the same brains that underwent thallium staining. Control sections underwent Nissl staining and staining without the HCl treatment, which leads to the detection of endogenous zinc.

Results

Thallium autometallography applied to juvenile gerbils (body weights around 7 to 13 g) gave sufficient visualisation of cortical activity, including the spatial resolution of single cortical columns. Prominent activity in the primary auditory field (A1) and the anterior auditory field (AAF) were evident in thallium as well as in FDG labelled gerbils. Methodological restrictions arose neither from the small size of the hearts perfused (around 60 to 100 mg) nor by histology in premature brains. The allometric relation between heart weight and body weight changes during early development in favour of relative bigger hearts in juvenile gerbils and relative small hearts in adults (Figure 1 [Fig. 1]).

Discussion

Sufficient perfusions do not interfere with the small size of hearts in juveniles, since the allometric relation between heart weight and body weight in gerbils changes during development. In contrast to the histological proceeding used in adult rodents [2], Thallium autometallography staining must be modified in juvenile animals. To relate labelling intensity of neuronal activity to body weight, thallium concentration was relatively enhanced in subadult Meriones unguiculatus (P 11-P 17), to face higher metabolic and relative greater heart weight as well as related blood volume during development. Therefore perfusion and histological proceeding in Thallium autometallography staining experiments in juvenile gerbils before onset of hearing could be continued successfully.

Ongoing research should allow the visualization of the neuronal activity with cellular resolution in large populations of neurons including some glial cells. The quality of the method can be increased by the use of glutaraldehyde to increase solution and minimize artefacts during section [2].

First experiments proved that the Thallium method could also be combined with behavioural experiments, e.g. auditory discrimination in the shuttle box and auditory stimulation with natural vocalisation during open field experiments. Yet it could not be clarified why superficial regions of the auditory cortex showed increased staining, which may point to enhanced activity within this regions during early sensory development.

Compared with the 2-fluoro-2-deoxyglucose method, Goldschmidt staining with Thallium provide a cheaper, faster and more robust method that gives a better solution to map neural activity. Radioactive substances are not involved, a fact reducing the dangerousness of activity labelling. The combined use of Goldschmidt staining and juvenile gerbils as an animal model for sensory development may lead to new insight on the early plasticity of auditory development.


References

1.
Stuermer I, Scheich H. Early unilateral auditory deprivation increases 2-deoxyglucose uptake in contralateral auditory cortex of juvenile Mongolian gerbils. Hear Res. 2000;146(1-2):185-99.
2.
Goldschmidt J, Zuschratter W, Scheich H. High-resolution mapping of neuronal activity by thallium autometallography. Neuroimage. 2004;23(2):638-47.
3.
Danscher G. Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electronmicroscopy. Histochemistry. 1981;71(1):1-16.