2 RATIONALE AND AIMS OF THE STUDY
5.4 Possible functional significance of the structural aberrations in TNR deficient mice
Behavioural analyses have revealed that adult TNR-deficient mice have motor deficits and that their neocortex is hyperexcitable. The results of this study show that TNR deficiency leads to aberration in subpopulations of inhibitory neurons in a region- and age-specific fashion. Reduced numbers of calretinin-positive interneurons at adult age might contribute, in an as to yet unknown way, to the dysbalance between excitation and inhibition in the hippocampus and neocortex of TNR deficient mice. The alterations in the size of the parvalbumin- and calretinin-positive interneuron populations in older age might be consequences of functional and structural deficits in these neuronal ensembles already present during adulthood but not detectable with the methods applied here. Future studies combining structural and functional analyses of the same animals may provide valuable new insight into pathophysiological mechanisms underlying brain dysfunctions.
6 SUMMARY
Tenascin-R (TNR) is an extracellular matrix glycoprotein belonging to the tenascin family of proteins. It is expressed by oligodendrocytes, interneurons and motoneurons in the central nervous system. In the adult mouse brain, TNR is found accumulated in perineuronal nets around motoneurons and interneurons and at nodes of Ranvier of myelinated axons. TNR is been implicated in cell adhesion, neurite outgrowth, modulation of ion channels and receptor functions, synaptic plasticity and learning. Constitutive ablation of TNR in mice causes behavioural and physiological abnormalities and impairment of motor coordination.
The morphological substrates of the deficits remain largely unknown.
The aim of this study was to analyse quantitatively neuronal and glial cell populations in the motor and sensory cortices of TNR-deficient mice. Densities and numbers per cortical column (cortical tissue under a unit of cortical surface) of immunohistochemically identified major cell types (neurons, neuronal subpopulations, astrocytes, oligodendrocytes and microglia) were stereologically assessed in TNR-deficient mice (TNR-/-) and wild-type (TNR+/+) littermates studied at ages of 5 and 18 months, i.e. adult and old age, respectively.
No differences between TNR-/- and TNR+/+ mice at both ages were found for cortical thickness, numbers of all cells and all neurons as well as for oligodendrocytes, astrocytes and microglial cells in both cortical areas. TNR deficiency had an cortical region-specific and age-related impact on the size of subpopulations of interneurons. Calretinin-positive interneurons were already significantly lower in numbers in adult TNR-/- mice compared to wild-type littermates (loss of about –20% in both cortical areas). Calbindin-positive interneuron numbers were normal in adult but reduced in old TNR-/- mice (-23%
and -29% compared to TNR+/+ mice in the motor and sensory cortex, respectively). Finally, age-related loss of parvalbumin-positive interneurons observed in TNR+/+ mice was found to be attenuated in the motor but not the sensory cortex of old TNR-/- animals.
Comparisons of old and adult mice revealed age-related but genotype-independent loss of excitatory neurons, inhibitory parvalbumin-positive
cortical thickness in the motor area of the neocortex. The magnitude of these age-related changes was between 18% and 44% compared with adult mice.
Interestingly, the sensory cortex was less severely affected by aging compared to the motor area in animals of both genotypes: small reduction in the numbers of all neurons (-10% in both genotype groups) and a moderate decrease in the numbers of parvalbumin-positive interneurons (-20% and –31% in TNR+/+ and TNR-/- mice, respectively).
The results of this study show that constitutive ablation of TNR does not influence the formation and maintenance throughout life of excitatory neuronal and glial cell populations in the neocortex. Affected by the mutation are subpopulation of interneurons with different functional characteristics and connectivity which may contribute to the previously observed functional deficits in the TNR deficient mouse. Ageing, independent of genotype, appears to influence the motor cortex more than the sensory cortical area. The finding of different degrees of neuronal loss in different neocortical areas is compatible with the differential age-related decline in different cognitive, motor and sensory functions.
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8 ABBREVATIONS
Ab Antibodies
AEPs Auditory evoked potentials
CA Cornu ammon
CaCl2 Calcium chloride
CHL1 Close homologue of L1
cm centimetres
cm² Square centrimetres
cm³ Cubic centrimetres
CNPase 2’, 3’-Cyclic Nucleotide 3’-Phosphodiesterase
CNS Central nervous system
ECM Extracellular matrix
EEG Electroenzephalogramm
e.g. example given
g gramm
GABA Gamma amino bytric acid
gt genotype
Hz Hertz
i.e. id est (that is)
Ig Immunglobulin
kD kilodalton
KO Knockout
l litre
LTP Long term potentiation
M Motor cortex
M molar
m metre
mm milimetre (Metre x 10-3) ml mililitre (Litre x 10-3)
mo month
ms milisecond (Second x 10-3) NaOH Sodium hydroxide solution
NaN3 Sodium azide
NeuN Neuron specific nuclear antigen
nm nanometres
PBS Phosphate Buffered Saline
pH p(otential) of H(ydrogen), the logarithm of the reciprocal of hydrogen-ion concentration in gram atoms per litre PNS Peripheral nervous system
PPI Prepulse inhibition
PSA Poly sialic acid
PV Parvalbumin
RT Room temperature
S Second
S Sensory Cortex
WT wildtype
w/v weight per volumn
ZMNH Zentrum für Molekulare Neurobiologie Hamburg
% Percent
µ Micro (10-6)
µg Microgramm (gramm x 10-6) µl Microlitre (Litre x 10-6)
°C Grad Celsius
9 ACKNOWLEDGEMENT/DANKSAGUNG
Die Arbeit wurde am Institut für Biosynthese neuraler Strukturen am Zentrum für molekulare Neurobiologie Hamburg (ZMNH) angefertigt. Bedanken möchte ich mich bei Frau Prof. Dr. Melitta Schachner, die diese Arbeit, als Doktormutter und Direktorin des Institutes, möglich gemacht hat.
Besonders bedanken möchte ich mich bei meinem Betreuer Herrn Dr. Andrey Irintchev, der mir jederzeit für Fragen aller Art zur Verfügung stand und keine Mühe scheute, mir bei dieser Arbeit in jeglicher Hinsicht zu helfen.
Ebenfalls möchte ich mich herzlich bei Frau Emanuela Szpotowicz für das Schneiden und Färben des Mausgewebes bedanken.
Mein besonderer Dank gilt auch Herrn Dr. Gheorghe Tonndorf, der mir helfend zur Seite stand und mich in vielerlei Hinsicht motiviert hat.
Nicht zu vergessen, möchte ich mich bei meinen Eltern Herrn Frank Salis und Frau Anita Salis und bei meiner Schwester Malinde Salis bedanken, die mich über die gesamte Zeit mit Interesse begleitet haben.
10 CURRICULUM VITAE
Personal Data
Family Name Salis
First Name Nadine
Date of Birth 09.07.1977
Place of Birth Hamburg, Germany
Nationality German
Confession Protestant
Education
1984-1988 Grundschule Egenbüttel, Rellingen, Germany
1988-1997 Wolfgang-Borchert-Gymnasium, Halstenbek, Germany 1997-2000 Apprenticeship in foreign trade
Nordmann, Rassmann GmbH & Co., Hamburg, Germany
2000-2006 Regular student of medicine Universität Hamburg, Germany