- Email: [email protected]
NFH-LacZ Transgenic Mice: Regional Brain Activity of Cytochrome Oxidase
Experimental Neurology 177, 521–530 (2002) doi:10.1006/exnr.2002.7972
NFH-LacZ Transgenic Mice: Regional Brain Activity of Cytochrome Oxidase C. Strazielle,* ,† ,1 M. Dubois,* J. Eyer,‡ and R. Lalonde* ,§ *Universite´ de Rouen, Faculte´ des Sciences, Laboratoire de Neurobiologie de l’Apprentissage, UPRES PSY.CO-EA 1780, 76821 MontSaint-Aignan Cedex, France; †Universite´ Henri Poincare´ Nancy I, Faculte´ de Me´decine, Service de Microscopie Electronique, 54500 Vandoeuvre-les-Nancy, France; ‡Universite´ d’Angers, Laboratoire de Neurobiologie et Transge´ne`se, UPRES-EA 3143, Baˆtiment Monteclair, CHU, 49033 Angers, France; and §CHUM/Hoˆtel-Dieu, Universite´ de Montre´al, Service de Neurologie, Montre´al, Que´bec, Canada H2W 1T8 Received July 19, 2001; accepted May 16, 2002
Expression of the NFH-LacZ fusion protein in transgenic mice causes an early accumulation of neurofilament proteins in the cell bodies of neurons, as well as a reduction of motor neuron axonal caliber and Purkinje cell number in the cerebellum. Young (3 month old) and older (12–20 months) NFH-LacZ transgenic mice were compared to normal controls for regional brain metabolism, as assessed by cytochrome oxidase (CO) activity. Irrespective of age, CO activity was reduced in three cerebellar-related regions of NFH-LacZ transgenic mice: (1) the lateral reticular nucleus, (2) the parvicellular red nucleus, and (3) the superior colliculus, possibly as a secondary consequence of cerebellar Purkinje cell histopathology. Aged NFH-LacZ mice had lower CO activity relative to either agematched controls or young transgenic mice in the following regions: the motor nucleus of the vagus nerve, the trapezoid nucleus, the subiculum, the motor cortex, the superior olive, and the lateral dorsal thalamus. These results indicate regional and age-selective deficits of brain metabolism in a transgenic model with neurofilament maldistribution. © 2002 Elsevier Science (USA)
Key Words: neurofilament; metabolic activity; spinal cord; cerebellum; amyotrophic lateral sclerosis.
INTRODUCTION
Three protein subunits have been identified in neurofilaments, varying in terms of molecular weight and of the timing of their expression during development: low (NFL), medium (NFM), and high (NFH). Integrated in the neuronal cytoskeleton, NFL and NFM are coexpressed during axonal growth while NFH is expressed at a later stage, mostly postnatally, when axons reach their target (4, 20). To whom reprint requests should be addressed. Fax: ⫹33 (0)3 83 68 29 81. E-mail: [email protected]. 1
In order to evaluate the role of those proteins in neural function, a transgenic mouse with a fusion gene between NFH and -galactosidase was created (10, 11, 38). The encoded fusion protein includes a truncated form of NFH and the complete -galactosidase. By comparison to littermate controls of the B6C3 strain, the neuronal cell bodies of the transgenic mouse develop Lewy body-like inclusions composed of massive filamentous aggregates including all three neurofilament subunits in addition to the NFH-LacZ fusion protein. Neurofilament-rich inclusions have also been observed in different lines of transgenic mice with insertion of the human NFH gene (5, 6). The inclusions in NFH-LacZ mice may compromise the viability of affected neurons, because cerebellar Purkinje cells containing type II inclusions which accumulate mitochondria and other organelles in cell bodies degenerate by 23% at 12 months of age and by 60% at 18 months of age (38). In contrast, no change in cell number was discerned in the CA1 subregion of the hippocampus in which perikaryal aggregates were devoid of cellular organelles (type I). Besides these neurofilament inclusions, NFL and NFM levels are decreased in cell bodies of NFH-LacZ transgenic mice, while NFH levels remain the same (38). However, NFH, normally the most phosphorylated among the three neurofilament subunits (42), is maintained in a hypophosphorylated state in these mice. The number of axonal neurofilaments is diminished in the peripheral and central nervous system (10) and a resulting reduction of the diameter of axons is detectable by Postnatal Day 5. However, peripheral axons are hypermyelinated and contain a higher density of microtubules, whereas central axons, also developing small calibers, present a proportionally reduced thickness of their myelin sheet (10). Neuronal conduction is slowed down by 50%, due mainly to the axonal caliber reduction. Nevertheless, NFH-LacZ transgenic mice are viable; they can reproduce and reach at least 20 months
521 ©
0014-4886/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
522
STRAZIELLE ET AL.
of age, although motor coordination deficits were discernable in young NFH-LacZ transgenic mice and were accentuated with aging (24). In the present study, the functional consequences of the redistribution of neurofilaments were examined by measuring regional brain metabolism. Because of the neuropathology in motor neurons and Purkinje cells occurring at different periods, the effects of the transgene were evaluated at a young age (3 months) and at a later age (12–20 months). Regional brain metabolism was assessed by means of cytochrome oxidase (CO) histochemistry (40, 41). CO is a mitochondrial enzyme essential for oxidative phosphorylation, whose activity is correlated with neuronal activity (41) and with other indices of cerebral metabolism (18). Regional metabolism has been shown to be altered in specific brain regions of ataxic mice with spontaneous mutations (23, 33). MATERIALS AND METHODS
Animals. Four groups of NFH-LacZ transgenic mice and controls (n ⫽ 10 for each group; N ⫽ 40) of the B6C3 strain (line 44A) were bred at Angers and sent to Rouen for behavioral evaluations. The groups were matched for sex and age (either at 3 or at 12–20 months) and kept in a room with a light-dark cycle of 14/10 h (lights on at 7:00), containing cages of 4 to 5 mice with woodchip bedding. Food and water were available at all times. The experimental design was in accordance with guidelines for the care and use of animals at each institution. Neurobehavioral performances, in particular motor activity and coordination, spatial learning, and exploratory behavior, were evaluated during a period of 16 days (24) before the assessment of CO histochemistry in Nancy. Tissue preparation. Two weeks after the termination of the behavioral studies, the animals were killed in the morning by decapitation and the brain was rapidly removed, frozen in ⫺40°C N-methylbutane, and stored at ⫺80°C. Subsequently, the brains were serially cut into 20-m-thick coronal sections at ⫺12°C with a cryostat, and the sections mounted on gelatinchrome alum-coated slides and stored at ⫺80°C until processing. CO histochemistry. CO histochemistry was performed simultaneously on two series of sections from each brain and several complete sets of standards, according to the protocol of Wong-Riley (40), slightly modified in the postincubation period (33). Slides were incubated in the dark for 75 min at 37°C in a solution of 0.1 M phosphate buffer (pH 7.4) containing 50 mg DAB, 20 mg horse-heart cytochrome c, 4 g sucrose, and 18 mg catalase per 90 ml, stirred continously. The slides were washed in cold buffer (4°C) for 5 min and immersed in a 10% buffered formalin solution for 30 min. The slides were then washed a second time in
buffer at room temperature (2 ⫻ 5 min), dehydrated in successive ethanol and xylene baths, and coverslipped with eukitt. Validation of the method was assured by the absence of the CO reaction product when DAB was removed or when 0.01 M potassium cyanide was added. Preparation of CO standards. To prepare standards for CO activity, cylindrical microtubes, filled by whole brain homogenates, were frozen and kept at ⫺80°C until cut in the cryostat at the same time as the brain sections. Sections 10, 20, and 40 m in thickness were used to cover the range of activity measured in the different structures by histochemistry. This standardization method has been previously validated by Gonzalez-Lima and Jones (16). Under our experimental conditions, the intensity of the staining was proportional to the thickness of the standard sections (r ⫽ ⫹0.995). The actual specific CO activity of these homogenates was measured by spectrophotometry (Uvikon 930, Kontron Instruments, Strasbourg, France) at 500 nm, using the method of Hess and Pope (19) modified as described by Strazielle et al. (33). The signal recorded was proportional to the amount of homogenate incubated and fully inhibited by potassium cyanide. The specific activity measured (30.51 mol/min/g of tissue) remained constant in ⫺80°C frozen standards for several months. Densitometric analysis. The CO histochemical staining intensity was quantified by densitometric analysis of the sections with a BIOCOM computer-assisted image analysis system (Les Ulis, France) and standards were used to convert optical density levels into enzymatic activity (in mol/min/g of tissue). Staining with a 1% methylene blue solution was performed on the adjacent slides for the purpose of delimiting precisely the different regions and subregions, and detecting possible cell defects or alterations, as well as regional atrophy in the transgenic mice. The entire brain could be observed on two slides, with the serial sections separated by 250-m intervals and the different regions measured were identified by means of the Franklin and Paxinos mouse atlas (14). Multiple optical density readings of CO labeling were made by a single operator under blinded conditions at magnifications ranging from 20 to 200⫻, depending on the heterogeneity of the tissue and the precision of the measures, from regional to cellular levels. To avoid contamination of adjacent structures and to obtain a homogeneous evaluation, measures with the same surface area for each site were obtained for each animal. Optical density readings were also performed on methylene blue-stained sections in some regions with altered metabolism. In addition, Purkinje cells were counted, known to be reduced during aging and some mutations (38), as well as in red nucleus, where only the cells of the magnocellular part of the red nucleus could be counted because of their large size. For the cerebellar cortex, the Purkinje cells of the simple lobule were manually counted on images magnified 100-fold,
523
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
TABLE 1 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Cerebellum of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control
NFH
Region
Young
Old
Young
Old
Granular layer Purkinje cells # Molecular layer # Fastigial Interpositus Dentate #
33.0 (1.2) 38.2 (1.4) 35.6 (1.1) 25.0 (1.0) 31.1 (1.4) 34.9 (0.9)
36.0 (1.7) 46.8 (1.5) 40.6 (2.0) 26.7 (1.0) 33.5 (1.1) 34.1 (1.1)
34.1 (1.1) 37.6 (1.6) 36.3 (1.5) 24.6 (1.0) 32.1 (1.1) 38.4 (1.4)
32.1 (1.2) 47.8 (2.7) 38.3 (1.4) 26.5 (0.9) 31.8 (1.1) 34.4 (0.9)
Note. CO activity in Purkinje cell was measured at the 200⫻ magnification level. # Age effect, P ⬍ 0.05.
for a unit length of 1000 m, defined by means of an IBM-compatible computer monitor. Similarly, the cells of the magnocellular red nucleus were counted for a comparatively identical surface area (0.04 mm 2) in the four groups of mice. Positive counts were recorded when the cell size was sufficient and the nucleolus was plainly visible. Statistical analyses. A factorial design comprising 2 ⫻ 2 analyses of variance (ANOVAs) was used, with the presence or absence of the transgene as one factor and the age of the animals as the second factor. Whenever the interaction was significant, two-tailed unpaired t tests were conducted in order to specify which group pairs differed significantly. RESULTS
CO activity in the entire brain of the four groups of mice is indicated in Tables 1–5. Because the intragroup variability in many brain regions was low, significant intergroup effects emerged even with relatively small changes in mean values. Transgene Effects The CO activity of NFH-LacZ mice was decreased in three brain stem regions: the parvicellular part of the red nucleus [F(1, 33) ⫽ 6.60, P ⬍ 0.05; Fig. 1], the lateral reticular nucleus [F(1, 30) ⫽ 4.73, P ⬍ 0.05], and the superior colliculus [F(1, 36) ⫽ 11.02, P ⬍ 0.01].
36) ⫽ 6.73, P ⬍ 0.05], as well as at the intracellular level of the cerebellar Purkinje cell [F(1, 36) ⫽ 25.54, P ⬍ 0.001], where enzymatic activity was measured at 200⫻ magnification (Fig. 2). On the contrary, CO activity in older mice was decreased in the cerebellar dentate nucleus [F(1, 35) ⫽ 4.63, P ⬍ 0.05], in the external cuneate nucleus [F(1, 33) ⫽ 4.33, P ⬍ 0.05], and in the intracellular compartment of the magnocellular part of the red nucleus measured at 200⫻ magnification [F(1, 34) ⫽ 12.47, P ⬍ 0.01]. Transgene ⫻ Age Interactions Interactions between the transgene factor and the aging factor occurred in six brain regions, namely three brain stem structures, the motor nucleus of the vagus nerve [F(1, 36) ⫽ 4.72, P ⬍ 0.05], the superior olive [F(1, 35) ⫽ 4.76, P ⬍ 0.05], and the trapezoid nucleus [F(1, 35) ⫽ 5.48, P ⬍ 0.05]; and three forebrain structures, the subiculum [F(1, 29) ⫽ 6.62, P ⬍ 0.05], the lateral dorsal nucleus of the thalamus [F(1, 34) ⫽ 5.01, P ⬍ 0.05], and the motor cortex [F(1, 24) ⫽ 4.89, P ⬍ 0.05]. Post hoc t tests revealed that the CO activity of aged controls was higher than that of young controls in the trapezoid nucleus [P ⬍ 0.05], the subiculum [P ⫽ 0.05], and the motor cortex [P ⫽ 0.05]. Although the same tendency was found for the motor nucleus of the vagus nerve, the result did not reach significance [P ⫽ 0.06]. In the superior olive and the lateral dorsal nucleus of the thalamus, the CO activity of older NFH-LacZ mice was lower than that of young NFH-LacZ mice [P ⬍ 0.05 and P ⬍ 0.01, respectively].
Age Effects Age-related changes in CO activity were found in seven brain regions. The entorhinal cortex was the only hypermetabolic region of the forebrain [F(1, 33) ⫽ 7.13, P ⬍ 0.05]. In the hindbrain, an age-related increase of CO activity was found in the molecular layer of the cerebellum [F(1, 36) ⫽ 5.13, P ⬍ 0.05], in the white matter of the cerebellar hemisphere [F(1,
Methylene Blue Staining For the Purkinje cell number/1000 m of length, counted in the cerebellar simple lobule, the gene ⫻ age interaction was significant [F(1, 33) ⫽ 4.50, P ⬍ 0.05]. The means ⫾ SEM were as follows: 42.1 ⫾ 1.4 for young controls, 37.1 ⫾ 1.3 for young NFH-LacZ, 38.5 ⫾ 0.9 for aged controls, and 28.1 ⫾ 1.5 for aged
524
STRAZIELLE ET AL.
TABLE 2 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Brain Stem of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control
NFH
Region
Young
Old
Young
Old
Subst nigra-compacta Subst nigra-reticulata Ventral tegmentum Periaqueductal grey Dorsal raphe n Medial raphe n Red n-magnocellular # Red n-parvicellular* Interpeduncular n Superior colliculus* Inferior colliculus Medial pontine Lateral pontine Gigantocellular ret n Paramedial ret n Lateral ret n* Ret tegmental n Trigeminal motor n Trigeminal spinal n Facial motor n Motor n vagus° Hypoglossal n Cochlear n Medial vestibular-pc Medial vestibular-mc Lateral vestibular n Superior vestibular n Spinal vestibular n Inferior olive Superior olive° Locus coeruleus Trapezoid n° Parabrachial n Area postrema N tractus solitarius External cuneate n #
27.4 (1.3) 26.8 (1.3) 18.4 (1.0) 28.1 (0.8) 31.5 (1.1) 29.3 (0.8) 54.3 (1.6) 27.0 (1.0) 49.5 (1.9) 29.5 (0.9) 36.2 (1.4) 27.0 (1.4) 41.8 (1.5) 29.6 (0.9) 23.1 (1.0) 35.1 (0.8) 30.4 (1.3) 33.9 (1.1) 36.4 (0.9) 39.4 (1.1) 27.8 (1.2) 40.2 (1.1) 42.2 (1.6) 46.5 (2.1) 35.5 (2.6) 26.9 (1.3) 26.7 (0.9) 25.0 (1.4) 52.8 (1.5) 36.4 (1.0) 28.5 (1.3) 28.8 (0.4) 28.8 (0.9) 24.4 (1.3) 26.6 (1.0) 37.2 (1.1)
27.8 (1.2) 25.5 (1.5) 19.1 (1.8) 27.7 (1.6) 32.9 (1.2) 31.0 (1.1) 49.7 (1.9) 27.7 (1.4) 51.4 (2.6) 30.8 (1.2) 36.8 (1.9) 28.9 (1.1) 43.9 (1.6) 30.2 (1.4) 24.8 (1.8) 35.9 (1.5) 30.9 (1.0) 33.9 (1.1) 36.7 (1.3) 40.0 (1.2) 31.5 (1.4) 42.1 (1.6) 40.8 (1.5) 48.6 (2.1) 36.8 (1.6) 25.1 (1.4) 26.6 (1.5) 28.3 (1.6) 51.3 (1.6) 38.8 (1.6) 28.3 (2.0) 32.0 (1.1) 29.5 (1.4) 26.3 (1.0) 29.2 (1.4) 35.8 (1.5)
24.9 (0.7) 24.2 (1.2) 17.7 (1.1) 28.0 (1.1) 32.6 (1.2) 30.9 (1.3) 53.4 (1.7) 25.5 (0.6) 46.8 (1.7) 26.2 (0.7) 31.7 (2.0) 28.4 (1.8) 42.4 (2.2) 28.1 (0.8) 21.9 (0.8) 33.8 (1.3) 30.9 (1.8) 33.7 (1.3) 34.2 (1.3) 37.6 (1.0) 29.4 (0.9) 42.3 (1.2) 41.8 (0.9) 47.4 (2.3) 33.8 (1.4) 27.1 (1.0) 27.0 (1.0) 25.8 (1.2) 51.9 (1.9) 40.2 (0.6) 30.5 (1.3) 30.4 (1.3) 30.0 (1.2) 23.3 (0.7) 28.6 (1.0) 37.2 (0.6)
28.1 (1.1) 25.0 (1.1) 20.7 (1.0) 27.2 (1.0) 32.1 (0.9) 28.8 (1.0) 46.4 (1.3) 24.4 (0.6) 50.3 (1.3) 27.9 (0.8) 37.9 (1.4) 27.1 (1.2) 43.6 (1.1) 29.0 (1.2) 21.3 (1.3) 31.1 (1.8) 29.4 (1.3) 33.5 (1.4) 36.2 (1.2) 37.5 (1.2) 28.3 (0.8) 39.2 (1.1) 40.1 (0.8) 46.4 (1.8) 35.0 (2.1) 24.4 (1.1) 27.1 (0.7) 25.1 (1.2) 49.7 (1.3) 37.9 (0.7) 28.5 (0.7) 28.4 (1.1) 29.2 (0.7) 25.1 (1.4) 27.1 (0.7) 34.3 (1.0)
Note. CO activity in the intracellular compartment of the magnocellular part of the red nucleus measured at the 200⫻ magnification level. n, nucleus; ret, reticular; subst, substantia; medial vestibular, parvicellular (pc), and mediocaudal (mc) parts. * Transgene effect, P ⬍ 0.05; #age effect, P ⬍ 0.05; °interaction effect, P ⬍ 0.05.
NFH-LacZ. Transgenic mice and older mice had a lower number of cells [P ⬍ 0.05] and the effect was especially potent in the aged transgenic mice. The mean ⫾ SEM number of cells/0.04 mm 2 measured in the magnocellular red nucleus was as follows: 16.3 ⫾ 0.6 for young controls, 14.2 ⫾ 1.3 for young NFH-LacZ, 14.2 ⫾ 0.8 for aged controls, and 12.4 ⫾ 1.1 for aged NFH-LacZ. The cell numbers were lower in transgenic mice [F(1, 29) ⫽ 4.30, P ⬍ 0.05] and in older mice [F(1, 29) ⫽ 4.42, P ⬍ 0.05], whereas the gene ⫻ age interaction was not significant [F(1, 29) ⫽ 0.02, P ⬎ 0.05]. On the contrary, no optical density variation was detected in the deep cerebellar nuclei and in the external cuneate nucleus [P ⬎ 0.05].
DISCUSSION
Cerebellum A 23– 60% reduction of Purkinje cell number was described in the entire cerebellar cortex of NFH-LacZ mice at 12–18 months but not at 6 months (38). The present study showed a Purkinje cell loss of approximatively 30% in the simple lobule of the aged NFHLacZ mice in comparison to age-matched controls. It remains to be determined whether other lobules of the cerebellar cortex degenerate as well and whether this degeneration occurs at a similar rate. In addition to being gene dependent, the Purkinje cell loss was also
525
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
TABLE 3 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Limbic System, Thalamus, and Subthalamic Nuclei of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control Region Hippocampus CA1 CA2-CA3 Dentate gyrus Subiculum° Thalamus nuclei Ventrolateral Ventroanterior Ventromedial Dorsomedial Midline Lateral geniculate Medial geniculate Ventroposterolateral Intralaminar Anterior Lateral dorsal° Posterior Subthalamic n Zona incerta Medial hypothalamus Mammillary bodies Amygdala Septum
NFH
Young
Old
Young
Old
24.4 (1.4) 27.9 (1.1) 25.8 (1.4) 30.3 (1.0)
27.1 (0.8) 29.6 (1.1) 28.1 (1.5) 34.1 (1.4)
25.8 (0.9) 27.3 (1.4) 25.0 (0.8) 33.1 (1.2)
26.2 (0.9) 29.3 (1.0) 25.9 (0.7) 30.4 (1.2)
30.7 (0.9) 23.5 (0.9) 27.0 (1.3) 35.0 (0.9) 28.0 (1.3) 29.3 (1.3) 29.8 (1.4) 29.9 (1.1) 27.7 (0.6) 31.6 (0.7) 31.6 (1.4) 28.2 (0.8) 33.3 (1.0) 30.9 (1.2) 25.8 (1.1) 37.9 (1.4) 26.3 (0.6) 27.4 (1.2)
32.2 (1.1) 23.5 (0.6) 28.6 (1.6) 33.7 (1.2) 29.3 (0.8) 30.4 (1.3) 33.3 (1.8) 30.5 (0.8) 27.7 (1.2) 33.3 (1.7) 31.8 (1.3) 29.4 (1.7) 36.0 (1.7) 34.4 (1.5) 28.5 (1.3) 38.2 (1.5) 29.2 (1.1) 29.9 (1.1)
32.6 (1.4) 24.2 (1.4) 27.8 (1.1) 34.8 (1.7) 28.2 (0.8) 32.7 (2.0) 29.9 (1.0) 29.8 (1.0) 28.6 (1.3) 32.6 (0.8) 35.0 (1.3) 27.7 (1.3) 33.4 (1.8) 31.1 (2.0) 26.9 (1.8) 36.4 (1.4) 28.1 (1.5) 30.0 (1.3)
32.1 (1.3) 23.6 (0.9) 29.5 (1.3) 34.9 (1.0) 29.6 (1.2) 27.9 (1.2) 29.9 (1.2) 30.5 (1.0) 28.5 (1.0) 31.5 (1.2) 29.5 (1.0) 26.4 (1.0) 31.8 (1.6) 28.9 (1.3) 24.3 (0.9) 34.7 (1.1) 27.6 (1.8) 29.2 (0.6)
° Interaction effect, P ⬍ 0.05.
age dependent, as the number of neurons was lower in aged mice. Despite this neuropathology, no transgenerelated change of CO activity was observed in the Purkinje cells and the cerebellar cortex. The type II neurofilament-rich inclusions observed in NFH-LacZ Purkinje cells (38), suspected of causing cell degeneration, did not seem to induce a direct CO alteration in the cerebellar cortex. An electron microscopic study should determine more precisely the possible effect of type II inclusions on Purkinje cell metabolic activity and viability. CO activity is reported in values relative to tissue weight. Because the Purkinje cell measure is intracellular, the values reflect the activity of the remaining cells. The normal CO activity values in NFH-LacZ mice mean that the remaining Purkinje cells are as metabolically active as the nondegenerated tissue of agematched controls. The unchanged CO activity observed in the NFH-LacZ deep cerebellar nuclei confirms this hypothesis. By contrast, Lurcher and staggerer ataxic mice characterized by massive Purkinje cell loss, despite an equal preservation of cerebellar cortical metabolic activity, displayed higher CO activity than controls in the deep nuclei (8, 33), perhaps indicating that the Purkinje cell loss in NFH-LacZ mice is insufficient for causing this elevation (24).
CO activity was increased in the molecular and Purkinje cell layers of the cerebellum in both groups of aged mice. A similar phenomenon was observed among the remaining ectopic Purkinje cells of staggerer mutants (8). This increase may reflect a functional compensation to aging Purkinje cells or the initial stage of a degenerative process. Cerebellar-Related Regions The CO activity of NFH-LacZ mice was decreased in three cerebellar-related regions: (1) the parvicellular part of the red nucleus (12, 21, 29), (2) the superior colliculus (12), and (3) the lateral reticular nucleus (31), perhaps as a result of the changes in Purkinje cell volume. In addition to cerebellar projections, these regions are interconnected and establish similar connections to other regions. The lateral reticular nucleus, providing a mossy fiber pathway to the cerebellar cortex and deep nuclei, receives its main afferent input from the frontoparietal sensorimotor cortex, the superior colliculus, the contralateral red nucleus, and the spinal cord (31, 32). Similarly, the superior colliculus is connected with the spinal cord, the motor cortex, and the cerebellar deep nuclei (1, 25, 27). Concerning the red nucleus, physiological and anatomical studies in
526
STRAZIELLE ET AL.
TABLE 4 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Telencephalon of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control Region Cerebral cortex Motor° Eye field Lateral prefrontal Cingulate Orbital Prelimbic Retrosplenial Anterior insular Anterior parietal Posterior parietal Entorhinal # Auditory Visual Piriform Olfactory tubercle Nucleus accumbens Striatum Dorsal lateral Dorsal medial Lateral pallidum #
NFH
Young
Old
Young
Old
27.7 (1.6) 28.2 (1.5) 31.2 (0.8) 29.7 (1.5) 29.9 (0.8) 27.1 (1.2) 33.0 (1.8) 30.0 (0.8) 28.8 (1.0) 31.2 (1.0) 28.1 (1.1) 29.6 (0.8) 30.9 (1.3) 31.0 (0.9) 34.5 (1.1) 36.6 (1.8)
32.1 (1.2) 31.2 (1.0) 30.8 (1.1) 32.7 (0.9) 30.1 (0.9) 28.2 (1.2) 34.4 (1.2) 30.2 (0.7) 31.9 (0.9) 31.2 (0.9) 30.7 (1.4) 30.2 (1.1) 32.7 (1.1) 32.5 (1.3) 36.0 (2.1) 40.0 (1.8)
30.2 (0.9) 29.0 (0.8) 31.3 (1.8) 31.7 (1.2) 31.0 (2.1) 27.0 (1.3) 30.8 (1.3) 31.3 (1.2) 29.6 (1.3) 31.2 (1.4) 26.7 (0.8) 29.0 (0.8) 31.4 (0.8) 30.5 (0.9) 34.2 (2.5) 36.8 (2.3)
29.5 (0.8) 29.5 (0.5) 30.6 (1.0) 31.5 (0.6) 28.9 (1.1) 27.7 (1.1) 33.9 (0.8) 30.5 (1.0) 30.0 (1.0) 33.1 (1.1) 30.2 (1.2) 29.2 (1.5) 30.0 (0.8) 30.9 (0.7) 36.5 (1.6) 38.6 (1.6)
29.5 (1.1) 27.6 (1.1) 21.1 (1.2)
32.0 (1.3) 29.8 (1.5) 24.0 (1.0)
30.8 (1.1) 28.5 (1.1) 20.6 (1.7)
29.6 (1.3) 26.9 (1.0) 21.6 (1.1)
Age effect, P ⬍ 0.05; °interaction effect, P ⬍ 0.05.
the rat (15, 29, 37) indicate that magno- and parvicellular parts of the red nucleus are controlled by cerebral as well as cerebellar influences. While the magnocellular red nucleus receives a predominant input from the interpositus nucleus (7, 9), the parvicellular part receives a predominant input from the dentate nucleus and the sensorimotor cortex (7, 17, 21). The two subregions differ as well in terms of their efferent connections. The magnocellular red nucleus projects mostly to the spinal cord (7, 9), while the parvicellular part projects preferentially to the inferior olive (22, 34).
These different neuroanatomical circuitries are summarized in Fig. 3. One of the possible reasons for the CO alterations in these regions is that metabolic depression occurred as a secondary consequence of cerebellar dysfunction. The CO activity of cerebellar-related pathways was also altered in Lurcher (33) and staggerer (8) mutants, but in the opposite direction (increased activity), as a possible result of the higher CO activity found in the deep cerebellar nuclei of these mice. By contrast, hypometabolism is an indication of cell stress rather than a sign of a compensatory action,
TABLE 5 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in White Matter Tracts of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control
#
NFH
Region
Young
Old
Young
Old
Optic nerve Medial lemniscus Fornix Mammillothalamic Nigrostriatal Medial longitudinal Cerebellar vermis Cerebellar hemisphere # Medial forebrain bundle
13.6 (0.6) 25.2 (1.2) 8.7 (0.9) 13.5 (1.0) 24.6 (1.0) 14.4 (1.5) 7.9 (0.7) 7.1 (0.6) 21.4 (1.1)
14.6 (1.3) 25.6 (1.1) 10.0 (0.6) 15.3 (1.3) 26.7 (1.7) 15.8 (0.9) 9.1 (0.6) 9.0 (0.4) 24.1 (2.0)
12.5 (1.3) 25.6 (1.2) 9.9 (1.1) 12.7 (0.9) 24.3 (1.4) 13.4 (1.0) 8.7 (0.6) 7.6 (0.5) 21.0 (1.5)
12.9 (1.5) 24.2 (1.4) 8.9 (0.8) 13.0 (1.0) 23.4 (1.0) 13.4 (1.1) 9.4 (0.6) 8.7 (0.7) 21.6 (0.9)
Age effect, P ⬍ 0.05.
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
527
FIG. 1. CO labeling of the red nucleus analyzed in young control (a) and in old NFH-LacZ (b) mice. Note the lower CO intensity of labeling in parvicellular part of the transgenic mouse region. (Scale bar ⫽ 100 m.) mc, magnocellular part of the red nucleus; PAG, periaqueductal grey nucleus; pc, parvicellular part of the red nucleus; RED, red nucleus.
indicating, therefore, that the type II neurofilamentrich inclusions in Purkinje cells may cause dysfunctions in connected structures. It is interesting to note that type II inclusions were observed among 60% of neocortical neurons in NFH-LacZ mice (38). Although it is not known whether the motor cortex is reduced in volume, it may be hypothesized that the lower CO activity found in the brain stem may also be a direct consequence of cortical afferent dysfunction. In addition, the reduced axonal calibers of the motor neurons in the spinal cord (10), associated with decreased neuronal conduction, might contribute as well to the metabolic alteration of these three brain stem motor regions. Because of the existence of projections from the superior colliculus and the red nucleus to the lateral reticular nucleus (30; Fig. 3), it is possible that CO alterations in the latter are caused not only by transsynaptic changes secondary to cerebellar damage but also by transsynaptic changes secondary to the depressed metabolic activity in both the superior colliculus and red nucleus. The reduced CO activity in the superior colliculus may also be due to the decreased optic nerve caliber in these mice (10), but this hypothesis is not supported by the lack of CO activity changes in the optic nerve, an area with few mitochondria.
In conclusion, despite the fact that NFH is expressed in most neurons of the central and peripheral nervous system (20), CO alterations in NFH-LacZ mice were limited to cerebellar- or cortical-related regions in the brain stem, resulting in selective deficits of motor coordination and visuomotor guidance while swimming toward a visible goal (24). In particular, low CO activity in the lateral reticular nucleus was associated with poor motor performance and low CO activity in the superior colliculus was associated with poor visuomotor performance (24). These results emphasize the functional consequence of cerebellar-related structures in this form of transgene expression. The Accentuated Transgene Effect during Aging The interactions between the transgene factor and the aging factor were significant in six brain regions, including brain stem (dorsal motor nucleus of the vagus nerve, superior olive, and trapezoid nucleus) and forebrain (lateral dorsal nucleus of the thalamus, subiculum, and motor cortex). In these regions, aging changed CO activity in an opposite way in NFH-LacZ mice as opposed to controls. In particular, CO activity increased in aged controls relative to young controls in
528
STRAZIELLE ET AL.
FIG. 2. Detail of the cerebellar cortex in young (a) and in aged (b) NFH-LacZ mice. A more intense CO labeling is observed in the molecular and Purkinje cell layers of the aged mouse in comparison to the young mouse; the arrows show the Purkinje cells. (Scale bar ⫽ 100 m.) Gr, granular layer; mol, molecular layer; Pj, Purkinje cell layer.
most of these regions. This was not the case in NFHLacZ mice, as their activity was decreased during aging. Previous experiments have indicated regionally selective changes in neurofilament levels during aging (39, 43). The present study indicates cell dysfunction in selective regions of aged NFH-LacZ mice as a possible result of the perikaryal neurofilament accumulations. Aging Effects in Cerebellum and Related Pathways Irrespective of the presence or the absence of the transgene, aged mice had lower CO activity in the dentate nucleus of the cerebellum, the magnocellular part of the red nucleus, and the external cuneate nucleus. On the contrary, CO activity was increased in the molecular layer and Purkinje cells of the cerebellum, in the lateral cerebellar white matter, and in the entorhinal cortex. Because of inhibitory GABAergic connections between Purkinje cells and the deep cerebellar nuclei (28), it is possible that the increased CO activity in the cerebellar cortex is directly responsible for the reduced CO activity in the dentate nucleus. Decreased CO activity in the magnocellular red nucleus may be a precursor to cell atrophy (36), as attested by the significant cell reduction found in the present study, but it may also be explained by the diminished metabolic activity in the dentate, as these projections are excitatory (37). Another possible expla-
nation of the selective regional hypometabolism found in the magnocellular red nucleus and the external cuneate nucleus, site of primary muscle afferents (2, 35), is based on their connections with motoneurons in the spinal cord. It has been shown that aging causes losses of motoneurons and external cuneate proprioceptive neurons in mice as early as 12 months (35). Because the magnocellular neurons give rise to most of the rubrospinal tract (7, 9), the CO activity reduction in this region could result from the age-related loss of motoneurons. CONCLUSION
The present study demonstrated that the neurofilament maldistribution seen in NFH-LacZ mice affects the metabolic activity of selective neuronal populations of the central nervous system. The entrapment of cellular organelles such as mitochondria in the perikaryon may affect neuronal metabolic activity and possibly its viability as a function of aging. Mitochondrial abnormalities characterize several neurodegenerative diseases with neurofilament inclusions, particularly amyotrophic lateral sclerosis (3, 13, 26). The NFH-LacZ mice will provide an opportunity for studying the role played by neurofilament inclusions on the mitochondrial compartment of the cells in the pathogenesis of the disease.
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
529
FIG. 3. Functional circuitries between brain stem structures mainly involved in motor control. Cerebellar afferents reach the cerebellar cortex, while efferents leave the cerebellum via the deep nuclei. Boxes illustrating the different structures appear more lighted when a significantly lower CO activity was observed in the NFH-LacZ mice (transgene effects). Double-way connections between structures are drawn in bold characters.
ACKNOWLEDGMENTS
6.
This work was supported by a grant from the Association Franc¸ aise de l’Ataxie de Friedreich (AFAF) to C.S. and R.L., and from the Association Franc¸ aise contre les Myopathies (AFM) to J.E.. We thank P. Robert and P. Chiron for technical assistance (animal facility and genotyping) at Angers.
7.
8.
REFERENCES 1.
2.
3.
4.
5.
Antonetty, C. M., and K. E. Webster. 1975. The organization of the spinotectal projections: An experimental study in the rat. J. Comp. Neurol. 163: 449 – 466. Bolton, P. S., and D. J. Tracey. 1992. The medullary relay from neck receptors to somatosensory thalamus in the rat: A neuroanatomical study. Exp. Brain Res. 88: 473– 484. Borthwick, C. M., M. A. Johnson, P. G. Ince, P. J. Shaw, and D. M. Turnbull. 1999. Mitochondrial enzyme activity in amyotrophic lateral sclerosis: Implications for the role of mitochondria in neuronal cell death. Ann. Neurol. 46: 787–790. Carden, M. J., J. Q. Trojanowski, W. W. Schlaepter, and V. M.-Y. Lee. 1987. Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7: 3489 –3504. Collard, J.-F., F. Coˆ te´ , and J.-P. Julien. 1995. Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. Nature 375: 61– 64.
9.
10.
11.
12.
13.
Coˆ te´ , F., J.-F. Collard, and J.-P. Julien. 1993. Progressive neuropathy in transgenic mice expressing the human neurofilament heavy gene: A mouse model of amyotrophic lateral sclerosis. Cell 73: 35– 46. Daniel, H., J. M. Billard, P. Angaut, and C. Batini. 1987. The interposito-rubrospinal system. Anatomical tracing of a motor control pathway in the rat. Neurosci. Res. 5: 87–112. Deiss, V., C. Strazielle, and R. Lalonde. 2000. Regional brain variations of cytochrome oxidase activity and motor co-ordination in staggerer mutant mice. Neuroscience 95: 903–911. Dekker, J. J. 1981. Anatomical evidence for direct fiber projections from the cerebellar nucleus interpositus to rubrospinal neurons. A quantitative EM study in the rat combining anterograde and retrograde intra-axonal tracing methods. Brain Res. 205: 229 –244. Eyer, J., and A. Peterson. 1994. Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament--galactosidase fusion protein. Neuron 12: 389 – 405. Eyer, J., D. W. Cleveland, P. C. Wong, and A. Peterson. 1998. Pathogenesis of two axonopathies does not require axonal neurofilaments. Nature 391: 584 –587. Faull, R. L. M., and J. B. Carman. 1978. The cerebellofugal projections in the brachium conjunctivum of the rat. I. The contralateral ascending pathway. J. Comp. Neurol. 178: 495–518. Forno, L. S. 1986. The Lewy body in Parkinson’s disease. Adv. Neurol. 45: 35– 43.
530 14. 15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
STRAZIELLE ET AL. Franklin, K. B. J., and G. Paxinos. 1997. The Mouse Brain in Stereotaxic Coordinates. Academic Press, New York. Giuffrida, R., G. Li Volsi, and V. Perciavalle. 1988. Influences of cerebral cortex and cerebellum on the red nucleus of the rat. Behav. Brain Res. 28: 109 –111. Gonzalez-Lima, F., and D. Jones. 1994. Quantitative mapping of cytochrome oxidase activity in the central auditory system of the gerbil: A study with calibrated activity standards and metalintensified histochemistry. Brain Res. 660: 34 –39. Gwyn, D. G., and P. A. Flumerfelt. 1974. A comparison of the distribution of cortical and cerebellar afferents in the red nucleus of the rat. Brain Res. 69: 130 –135. Harley, C. A., and C. H. Bielajew. 1992. A comparison of glycogen phosphorylase a and cytochrome oxidase histochemical staining in rat brain. J. Comp. Neurol. 322: 377–389. Hess, H. H., and A. Pope. 1953. Ultramicrospectrophotometric determination of cytochrome oxidase for quantitative histochemistry. J. Biol. Chem. 204: 295–306. Julien, J.-P., D. Meijer, J. Hurst, and F. Grosveld. 1986. Cloning and developmental expression of the murine neurofilament gene family. Mol. Brain Res. 1: 243–250. Kennedy, P. R., A. R. Gibson, and J. C. Houk. 1986. Functional and anatomic differentiation between parvicellular and magnocellular regions of red nucleus in the monkey. Brain Res. 364: 124 –136. Kennedy, P. R. 1990. Corticospinal, rubrospinal and rubroolivary projections: A unifying hypothesis. Trends Neurosci. 13: 474 – 479. Kre´ marik, P., C. Strazielle, and R. Lalonde. 1998. Regional brain variations of cytochrome oxidase activity and motor coordination in hot-foot mutant mice. Eur. J. Neurosci. 10: 2802– 2809. Lalonde, R., M. Dubois, C. Strazielle, and J. Eyer. 1999. Motor coordination and spatial orientation are affected by neurofilament maldistribution: Correlations with regional brain activity of cytochrome oxidase. Exp. Brain Res. 126: 223–234. Lee, H. S., R. J. Kosinski, and G. A. Mihailoff. 1989. Collateral branches of cerebellopontine axons reach the thalamus, superior colliculus or inferior olive: A double fluorescence and combined fluorescence-horseradish peroxidase study in the rat. Neuroscience 28: 725–735. Murayama, S., Y. Ookawa, H. Mori, I. Nakano, Y. Ihara, S. Kuzuhara, and M. Tomonaga. 1989. Immunocytochemical and ultrastructural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis. Acta Neuropathol. 78: 143–152. Myashita E., E. Keller, and H. Asuma. 1994. Input-output organization of the rat vibrissal motor cortex. Exp. Brain Res. 99: 223–232.
28. 29.
30.
31.
32.
33.
34.
35. 36. 37.
38.
39.
40.
41.
42.
43.
Oertel, W. H. 1993. Neurotransmitters in the cerebellum: Scientific aspects and clinical relevance. Adv. Neurol. 61: 33–75. Perciavalle, V., S. Berretta, and R. Raffaele. 1989. Projections from the intracerebellar nuclei to the ventral midbrain tegmentum in the rat. Neuroscience 29: 109 –119. Qvist, H., and E. Dietrichs. 1985. The projection from the superior colliculus to the lateral reticular nucleus in the cat as studied with retrograde transport of WGA-HRP. Anat. Embryol. 173: 269 –274. Rajakumar, N., A. W. Hrycyshyn, and B. A. Flumerfelt. 1992. Afferent organization of the lateral reticular nucleus in the rat: An anterograde tracing study. Anat. Embryol. 185: 25–37. Shokunbi, M. T., A. W. Hrycyshyn, and B. A. Flumerfelt. 1986. A horseradish peroxidase study of the rubral and cortical afferents to the lateral reticular nucleus in the rat. J. Comp. Neurol. 248: 441– 454. Strazielle, C., P. Kre´ marik, J.-F. Ghersi-Egea, and R. Lalonde. 1998. Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice. Exp. Brain Res. 121: 35– 45. Strominger, N. L., T. C. Truscott, R. A. Miller, and G. J. Royce. 1979. An autoradiographic study of the rubro-olivary tract in the rhesus monkey. J. Comp. Neurol. 183: 33– 46. Sturrock, R. R. 1989. Loss of neurons from the external cuneate of the ageing mouse brain. J. Anat. 163: 135–141. Sturrock, R. R. 1990. Age related changes in neuron number in the mouse red nucleus. J. Hirnforsch. 31: 399 – 403. Toyama, K., N. Tsukuhara, and M. Udo. 1967. Nature of cerebellar influences upon the red nucleus neurones. Exp. Brain Res. 4: 292–309. Tu, P.-H., K. A. Robinson, F. de Snoo, J. Eyer, A. Peterson, V. M.-Y. Lee, and J. Q. Trojanowski. 1997. Selective degeneration of Purkinje cells with Lewy body-like inclusions in aged NFHLacZ transgenic mice. J. Neurosci. 17: 1064 –1074. Van Der Zee, E. A., P. A. Naber, and J. F. Disterhoft. 1997. Age-dependent changes in the immunoreactivity for neurofilaments in rabbit hippocampus. Neuroscience 79: 103–116. Wong-Riley, M. T. T. 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171: 11–28. Wong-Riley, M. T. T. 1989. Cytochrome oxidase: An endogenous metabolic marker for neuronal activity. Trends Neurosci. 12: 94 –101. Xu Z., D. L.-Y. Dong, and D. W. Cleveland. 1994. Neuronal intermediate filaments: New progress on an old subject. Curr. Opin. Neurobiol. 4: 655– 661. Zhang, J.-H., S. Sampogna, F. R. Morales, and M. H. Chase. 1997. Age-related alterations in immunoreactivity of the midsized neurofilament subunit in the brainstem reticular formation of the cat. Brain Res. 769: 196 –200.
NFH-LacZ Transgenic Mice: Regional Brain Activity of Cytochrome Oxidase C. Strazielle,* ,† ,1 M. Dubois,* J. Eyer,‡ and R. Lalonde* ,§ *Universite´ de Rouen, Faculte´ des Sciences, Laboratoire de Neurobiologie de l’Apprentissage, UPRES PSY.CO-EA 1780, 76821 MontSaint-Aignan Cedex, France; †Universite´ Henri Poincare´ Nancy I, Faculte´ de Me´decine, Service de Microscopie Electronique, 54500 Vandoeuvre-les-Nancy, France; ‡Universite´ d’Angers, Laboratoire de Neurobiologie et Transge´ne`se, UPRES-EA 3143, Baˆtiment Monteclair, CHU, 49033 Angers, France; and §CHUM/Hoˆtel-Dieu, Universite´ de Montre´al, Service de Neurologie, Montre´al, Que´bec, Canada H2W 1T8 Received July 19, 2001; accepted May 16, 2002
Expression of the NFH-LacZ fusion protein in transgenic mice causes an early accumulation of neurofilament proteins in the cell bodies of neurons, as well as a reduction of motor neuron axonal caliber and Purkinje cell number in the cerebellum. Young (3 month old) and older (12–20 months) NFH-LacZ transgenic mice were compared to normal controls for regional brain metabolism, as assessed by cytochrome oxidase (CO) activity. Irrespective of age, CO activity was reduced in three cerebellar-related regions of NFH-LacZ transgenic mice: (1) the lateral reticular nucleus, (2) the parvicellular red nucleus, and (3) the superior colliculus, possibly as a secondary consequence of cerebellar Purkinje cell histopathology. Aged NFH-LacZ mice had lower CO activity relative to either agematched controls or young transgenic mice in the following regions: the motor nucleus of the vagus nerve, the trapezoid nucleus, the subiculum, the motor cortex, the superior olive, and the lateral dorsal thalamus. These results indicate regional and age-selective deficits of brain metabolism in a transgenic model with neurofilament maldistribution. © 2002 Elsevier Science (USA)
Key Words: neurofilament; metabolic activity; spinal cord; cerebellum; amyotrophic lateral sclerosis.
INTRODUCTION
Three protein subunits have been identified in neurofilaments, varying in terms of molecular weight and of the timing of their expression during development: low (NFL), medium (NFM), and high (NFH). Integrated in the neuronal cytoskeleton, NFL and NFM are coexpressed during axonal growth while NFH is expressed at a later stage, mostly postnatally, when axons reach their target (4, 20). To whom reprint requests should be addressed. Fax: ⫹33 (0)3 83 68 29 81. E-mail: [email protected]. 1
In order to evaluate the role of those proteins in neural function, a transgenic mouse with a fusion gene between NFH and -galactosidase was created (10, 11, 38). The encoded fusion protein includes a truncated form of NFH and the complete -galactosidase. By comparison to littermate controls of the B6C3 strain, the neuronal cell bodies of the transgenic mouse develop Lewy body-like inclusions composed of massive filamentous aggregates including all three neurofilament subunits in addition to the NFH-LacZ fusion protein. Neurofilament-rich inclusions have also been observed in different lines of transgenic mice with insertion of the human NFH gene (5, 6). The inclusions in NFH-LacZ mice may compromise the viability of affected neurons, because cerebellar Purkinje cells containing type II inclusions which accumulate mitochondria and other organelles in cell bodies degenerate by 23% at 12 months of age and by 60% at 18 months of age (38). In contrast, no change in cell number was discerned in the CA1 subregion of the hippocampus in which perikaryal aggregates were devoid of cellular organelles (type I). Besides these neurofilament inclusions, NFL and NFM levels are decreased in cell bodies of NFH-LacZ transgenic mice, while NFH levels remain the same (38). However, NFH, normally the most phosphorylated among the three neurofilament subunits (42), is maintained in a hypophosphorylated state in these mice. The number of axonal neurofilaments is diminished in the peripheral and central nervous system (10) and a resulting reduction of the diameter of axons is detectable by Postnatal Day 5. However, peripheral axons are hypermyelinated and contain a higher density of microtubules, whereas central axons, also developing small calibers, present a proportionally reduced thickness of their myelin sheet (10). Neuronal conduction is slowed down by 50%, due mainly to the axonal caliber reduction. Nevertheless, NFH-LacZ transgenic mice are viable; they can reproduce and reach at least 20 months
521 ©
0014-4886/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
522
STRAZIELLE ET AL.
of age, although motor coordination deficits were discernable in young NFH-LacZ transgenic mice and were accentuated with aging (24). In the present study, the functional consequences of the redistribution of neurofilaments were examined by measuring regional brain metabolism. Because of the neuropathology in motor neurons and Purkinje cells occurring at different periods, the effects of the transgene were evaluated at a young age (3 months) and at a later age (12–20 months). Regional brain metabolism was assessed by means of cytochrome oxidase (CO) histochemistry (40, 41). CO is a mitochondrial enzyme essential for oxidative phosphorylation, whose activity is correlated with neuronal activity (41) and with other indices of cerebral metabolism (18). Regional metabolism has been shown to be altered in specific brain regions of ataxic mice with spontaneous mutations (23, 33). MATERIALS AND METHODS
Animals. Four groups of NFH-LacZ transgenic mice and controls (n ⫽ 10 for each group; N ⫽ 40) of the B6C3 strain (line 44A) were bred at Angers and sent to Rouen for behavioral evaluations. The groups were matched for sex and age (either at 3 or at 12–20 months) and kept in a room with a light-dark cycle of 14/10 h (lights on at 7:00), containing cages of 4 to 5 mice with woodchip bedding. Food and water were available at all times. The experimental design was in accordance with guidelines for the care and use of animals at each institution. Neurobehavioral performances, in particular motor activity and coordination, spatial learning, and exploratory behavior, were evaluated during a period of 16 days (24) before the assessment of CO histochemistry in Nancy. Tissue preparation. Two weeks after the termination of the behavioral studies, the animals were killed in the morning by decapitation and the brain was rapidly removed, frozen in ⫺40°C N-methylbutane, and stored at ⫺80°C. Subsequently, the brains were serially cut into 20-m-thick coronal sections at ⫺12°C with a cryostat, and the sections mounted on gelatinchrome alum-coated slides and stored at ⫺80°C until processing. CO histochemistry. CO histochemistry was performed simultaneously on two series of sections from each brain and several complete sets of standards, according to the protocol of Wong-Riley (40), slightly modified in the postincubation period (33). Slides were incubated in the dark for 75 min at 37°C in a solution of 0.1 M phosphate buffer (pH 7.4) containing 50 mg DAB, 20 mg horse-heart cytochrome c, 4 g sucrose, and 18 mg catalase per 90 ml, stirred continously. The slides were washed in cold buffer (4°C) for 5 min and immersed in a 10% buffered formalin solution for 30 min. The slides were then washed a second time in
buffer at room temperature (2 ⫻ 5 min), dehydrated in successive ethanol and xylene baths, and coverslipped with eukitt. Validation of the method was assured by the absence of the CO reaction product when DAB was removed or when 0.01 M potassium cyanide was added. Preparation of CO standards. To prepare standards for CO activity, cylindrical microtubes, filled by whole brain homogenates, were frozen and kept at ⫺80°C until cut in the cryostat at the same time as the brain sections. Sections 10, 20, and 40 m in thickness were used to cover the range of activity measured in the different structures by histochemistry. This standardization method has been previously validated by Gonzalez-Lima and Jones (16). Under our experimental conditions, the intensity of the staining was proportional to the thickness of the standard sections (r ⫽ ⫹0.995). The actual specific CO activity of these homogenates was measured by spectrophotometry (Uvikon 930, Kontron Instruments, Strasbourg, France) at 500 nm, using the method of Hess and Pope (19) modified as described by Strazielle et al. (33). The signal recorded was proportional to the amount of homogenate incubated and fully inhibited by potassium cyanide. The specific activity measured (30.51 mol/min/g of tissue) remained constant in ⫺80°C frozen standards for several months. Densitometric analysis. The CO histochemical staining intensity was quantified by densitometric analysis of the sections with a BIOCOM computer-assisted image analysis system (Les Ulis, France) and standards were used to convert optical density levels into enzymatic activity (in mol/min/g of tissue). Staining with a 1% methylene blue solution was performed on the adjacent slides for the purpose of delimiting precisely the different regions and subregions, and detecting possible cell defects or alterations, as well as regional atrophy in the transgenic mice. The entire brain could be observed on two slides, with the serial sections separated by 250-m intervals and the different regions measured were identified by means of the Franklin and Paxinos mouse atlas (14). Multiple optical density readings of CO labeling were made by a single operator under blinded conditions at magnifications ranging from 20 to 200⫻, depending on the heterogeneity of the tissue and the precision of the measures, from regional to cellular levels. To avoid contamination of adjacent structures and to obtain a homogeneous evaluation, measures with the same surface area for each site were obtained for each animal. Optical density readings were also performed on methylene blue-stained sections in some regions with altered metabolism. In addition, Purkinje cells were counted, known to be reduced during aging and some mutations (38), as well as in red nucleus, where only the cells of the magnocellular part of the red nucleus could be counted because of their large size. For the cerebellar cortex, the Purkinje cells of the simple lobule were manually counted on images magnified 100-fold,
523
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
TABLE 1 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Cerebellum of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control
NFH
Region
Young
Old
Young
Old
Granular layer Purkinje cells # Molecular layer # Fastigial Interpositus Dentate #
33.0 (1.2) 38.2 (1.4) 35.6 (1.1) 25.0 (1.0) 31.1 (1.4) 34.9 (0.9)
36.0 (1.7) 46.8 (1.5) 40.6 (2.0) 26.7 (1.0) 33.5 (1.1) 34.1 (1.1)
34.1 (1.1) 37.6 (1.6) 36.3 (1.5) 24.6 (1.0) 32.1 (1.1) 38.4 (1.4)
32.1 (1.2) 47.8 (2.7) 38.3 (1.4) 26.5 (0.9) 31.8 (1.1) 34.4 (0.9)
Note. CO activity in Purkinje cell was measured at the 200⫻ magnification level. # Age effect, P ⬍ 0.05.
for a unit length of 1000 m, defined by means of an IBM-compatible computer monitor. Similarly, the cells of the magnocellular red nucleus were counted for a comparatively identical surface area (0.04 mm 2) in the four groups of mice. Positive counts were recorded when the cell size was sufficient and the nucleolus was plainly visible. Statistical analyses. A factorial design comprising 2 ⫻ 2 analyses of variance (ANOVAs) was used, with the presence or absence of the transgene as one factor and the age of the animals as the second factor. Whenever the interaction was significant, two-tailed unpaired t tests were conducted in order to specify which group pairs differed significantly. RESULTS
CO activity in the entire brain of the four groups of mice is indicated in Tables 1–5. Because the intragroup variability in many brain regions was low, significant intergroup effects emerged even with relatively small changes in mean values. Transgene Effects The CO activity of NFH-LacZ mice was decreased in three brain stem regions: the parvicellular part of the red nucleus [F(1, 33) ⫽ 6.60, P ⬍ 0.05; Fig. 1], the lateral reticular nucleus [F(1, 30) ⫽ 4.73, P ⬍ 0.05], and the superior colliculus [F(1, 36) ⫽ 11.02, P ⬍ 0.01].
36) ⫽ 6.73, P ⬍ 0.05], as well as at the intracellular level of the cerebellar Purkinje cell [F(1, 36) ⫽ 25.54, P ⬍ 0.001], where enzymatic activity was measured at 200⫻ magnification (Fig. 2). On the contrary, CO activity in older mice was decreased in the cerebellar dentate nucleus [F(1, 35) ⫽ 4.63, P ⬍ 0.05], in the external cuneate nucleus [F(1, 33) ⫽ 4.33, P ⬍ 0.05], and in the intracellular compartment of the magnocellular part of the red nucleus measured at 200⫻ magnification [F(1, 34) ⫽ 12.47, P ⬍ 0.01]. Transgene ⫻ Age Interactions Interactions between the transgene factor and the aging factor occurred in six brain regions, namely three brain stem structures, the motor nucleus of the vagus nerve [F(1, 36) ⫽ 4.72, P ⬍ 0.05], the superior olive [F(1, 35) ⫽ 4.76, P ⬍ 0.05], and the trapezoid nucleus [F(1, 35) ⫽ 5.48, P ⬍ 0.05]; and three forebrain structures, the subiculum [F(1, 29) ⫽ 6.62, P ⬍ 0.05], the lateral dorsal nucleus of the thalamus [F(1, 34) ⫽ 5.01, P ⬍ 0.05], and the motor cortex [F(1, 24) ⫽ 4.89, P ⬍ 0.05]. Post hoc t tests revealed that the CO activity of aged controls was higher than that of young controls in the trapezoid nucleus [P ⬍ 0.05], the subiculum [P ⫽ 0.05], and the motor cortex [P ⫽ 0.05]. Although the same tendency was found for the motor nucleus of the vagus nerve, the result did not reach significance [P ⫽ 0.06]. In the superior olive and the lateral dorsal nucleus of the thalamus, the CO activity of older NFH-LacZ mice was lower than that of young NFH-LacZ mice [P ⬍ 0.05 and P ⬍ 0.01, respectively].
Age Effects Age-related changes in CO activity were found in seven brain regions. The entorhinal cortex was the only hypermetabolic region of the forebrain [F(1, 33) ⫽ 7.13, P ⬍ 0.05]. In the hindbrain, an age-related increase of CO activity was found in the molecular layer of the cerebellum [F(1, 36) ⫽ 5.13, P ⬍ 0.05], in the white matter of the cerebellar hemisphere [F(1,
Methylene Blue Staining For the Purkinje cell number/1000 m of length, counted in the cerebellar simple lobule, the gene ⫻ age interaction was significant [F(1, 33) ⫽ 4.50, P ⬍ 0.05]. The means ⫾ SEM were as follows: 42.1 ⫾ 1.4 for young controls, 37.1 ⫾ 1.3 for young NFH-LacZ, 38.5 ⫾ 0.9 for aged controls, and 28.1 ⫾ 1.5 for aged
524
STRAZIELLE ET AL.
TABLE 2 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Brain Stem of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control
NFH
Region
Young
Old
Young
Old
Subst nigra-compacta Subst nigra-reticulata Ventral tegmentum Periaqueductal grey Dorsal raphe n Medial raphe n Red n-magnocellular # Red n-parvicellular* Interpeduncular n Superior colliculus* Inferior colliculus Medial pontine Lateral pontine Gigantocellular ret n Paramedial ret n Lateral ret n* Ret tegmental n Trigeminal motor n Trigeminal spinal n Facial motor n Motor n vagus° Hypoglossal n Cochlear n Medial vestibular-pc Medial vestibular-mc Lateral vestibular n Superior vestibular n Spinal vestibular n Inferior olive Superior olive° Locus coeruleus Trapezoid n° Parabrachial n Area postrema N tractus solitarius External cuneate n #
27.4 (1.3) 26.8 (1.3) 18.4 (1.0) 28.1 (0.8) 31.5 (1.1) 29.3 (0.8) 54.3 (1.6) 27.0 (1.0) 49.5 (1.9) 29.5 (0.9) 36.2 (1.4) 27.0 (1.4) 41.8 (1.5) 29.6 (0.9) 23.1 (1.0) 35.1 (0.8) 30.4 (1.3) 33.9 (1.1) 36.4 (0.9) 39.4 (1.1) 27.8 (1.2) 40.2 (1.1) 42.2 (1.6) 46.5 (2.1) 35.5 (2.6) 26.9 (1.3) 26.7 (0.9) 25.0 (1.4) 52.8 (1.5) 36.4 (1.0) 28.5 (1.3) 28.8 (0.4) 28.8 (0.9) 24.4 (1.3) 26.6 (1.0) 37.2 (1.1)
27.8 (1.2) 25.5 (1.5) 19.1 (1.8) 27.7 (1.6) 32.9 (1.2) 31.0 (1.1) 49.7 (1.9) 27.7 (1.4) 51.4 (2.6) 30.8 (1.2) 36.8 (1.9) 28.9 (1.1) 43.9 (1.6) 30.2 (1.4) 24.8 (1.8) 35.9 (1.5) 30.9 (1.0) 33.9 (1.1) 36.7 (1.3) 40.0 (1.2) 31.5 (1.4) 42.1 (1.6) 40.8 (1.5) 48.6 (2.1) 36.8 (1.6) 25.1 (1.4) 26.6 (1.5) 28.3 (1.6) 51.3 (1.6) 38.8 (1.6) 28.3 (2.0) 32.0 (1.1) 29.5 (1.4) 26.3 (1.0) 29.2 (1.4) 35.8 (1.5)
24.9 (0.7) 24.2 (1.2) 17.7 (1.1) 28.0 (1.1) 32.6 (1.2) 30.9 (1.3) 53.4 (1.7) 25.5 (0.6) 46.8 (1.7) 26.2 (0.7) 31.7 (2.0) 28.4 (1.8) 42.4 (2.2) 28.1 (0.8) 21.9 (0.8) 33.8 (1.3) 30.9 (1.8) 33.7 (1.3) 34.2 (1.3) 37.6 (1.0) 29.4 (0.9) 42.3 (1.2) 41.8 (0.9) 47.4 (2.3) 33.8 (1.4) 27.1 (1.0) 27.0 (1.0) 25.8 (1.2) 51.9 (1.9) 40.2 (0.6) 30.5 (1.3) 30.4 (1.3) 30.0 (1.2) 23.3 (0.7) 28.6 (1.0) 37.2 (0.6)
28.1 (1.1) 25.0 (1.1) 20.7 (1.0) 27.2 (1.0) 32.1 (0.9) 28.8 (1.0) 46.4 (1.3) 24.4 (0.6) 50.3 (1.3) 27.9 (0.8) 37.9 (1.4) 27.1 (1.2) 43.6 (1.1) 29.0 (1.2) 21.3 (1.3) 31.1 (1.8) 29.4 (1.3) 33.5 (1.4) 36.2 (1.2) 37.5 (1.2) 28.3 (0.8) 39.2 (1.1) 40.1 (0.8) 46.4 (1.8) 35.0 (2.1) 24.4 (1.1) 27.1 (0.7) 25.1 (1.2) 49.7 (1.3) 37.9 (0.7) 28.5 (0.7) 28.4 (1.1) 29.2 (0.7) 25.1 (1.4) 27.1 (0.7) 34.3 (1.0)
Note. CO activity in the intracellular compartment of the magnocellular part of the red nucleus measured at the 200⫻ magnification level. n, nucleus; ret, reticular; subst, substantia; medial vestibular, parvicellular (pc), and mediocaudal (mc) parts. * Transgene effect, P ⬍ 0.05; #age effect, P ⬍ 0.05; °interaction effect, P ⬍ 0.05.
NFH-LacZ. Transgenic mice and older mice had a lower number of cells [P ⬍ 0.05] and the effect was especially potent in the aged transgenic mice. The mean ⫾ SEM number of cells/0.04 mm 2 measured in the magnocellular red nucleus was as follows: 16.3 ⫾ 0.6 for young controls, 14.2 ⫾ 1.3 for young NFH-LacZ, 14.2 ⫾ 0.8 for aged controls, and 12.4 ⫾ 1.1 for aged NFH-LacZ. The cell numbers were lower in transgenic mice [F(1, 29) ⫽ 4.30, P ⬍ 0.05] and in older mice [F(1, 29) ⫽ 4.42, P ⬍ 0.05], whereas the gene ⫻ age interaction was not significant [F(1, 29) ⫽ 0.02, P ⬎ 0.05]. On the contrary, no optical density variation was detected in the deep cerebellar nuclei and in the external cuneate nucleus [P ⬎ 0.05].
DISCUSSION
Cerebellum A 23– 60% reduction of Purkinje cell number was described in the entire cerebellar cortex of NFH-LacZ mice at 12–18 months but not at 6 months (38). The present study showed a Purkinje cell loss of approximatively 30% in the simple lobule of the aged NFHLacZ mice in comparison to age-matched controls. It remains to be determined whether other lobules of the cerebellar cortex degenerate as well and whether this degeneration occurs at a similar rate. In addition to being gene dependent, the Purkinje cell loss was also
525
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
TABLE 3 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Limbic System, Thalamus, and Subthalamic Nuclei of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control Region Hippocampus CA1 CA2-CA3 Dentate gyrus Subiculum° Thalamus nuclei Ventrolateral Ventroanterior Ventromedial Dorsomedial Midline Lateral geniculate Medial geniculate Ventroposterolateral Intralaminar Anterior Lateral dorsal° Posterior Subthalamic n Zona incerta Medial hypothalamus Mammillary bodies Amygdala Septum
NFH
Young
Old
Young
Old
24.4 (1.4) 27.9 (1.1) 25.8 (1.4) 30.3 (1.0)
27.1 (0.8) 29.6 (1.1) 28.1 (1.5) 34.1 (1.4)
25.8 (0.9) 27.3 (1.4) 25.0 (0.8) 33.1 (1.2)
26.2 (0.9) 29.3 (1.0) 25.9 (0.7) 30.4 (1.2)
30.7 (0.9) 23.5 (0.9) 27.0 (1.3) 35.0 (0.9) 28.0 (1.3) 29.3 (1.3) 29.8 (1.4) 29.9 (1.1) 27.7 (0.6) 31.6 (0.7) 31.6 (1.4) 28.2 (0.8) 33.3 (1.0) 30.9 (1.2) 25.8 (1.1) 37.9 (1.4) 26.3 (0.6) 27.4 (1.2)
32.2 (1.1) 23.5 (0.6) 28.6 (1.6) 33.7 (1.2) 29.3 (0.8) 30.4 (1.3) 33.3 (1.8) 30.5 (0.8) 27.7 (1.2) 33.3 (1.7) 31.8 (1.3) 29.4 (1.7) 36.0 (1.7) 34.4 (1.5) 28.5 (1.3) 38.2 (1.5) 29.2 (1.1) 29.9 (1.1)
32.6 (1.4) 24.2 (1.4) 27.8 (1.1) 34.8 (1.7) 28.2 (0.8) 32.7 (2.0) 29.9 (1.0) 29.8 (1.0) 28.6 (1.3) 32.6 (0.8) 35.0 (1.3) 27.7 (1.3) 33.4 (1.8) 31.1 (2.0) 26.9 (1.8) 36.4 (1.4) 28.1 (1.5) 30.0 (1.3)
32.1 (1.3) 23.6 (0.9) 29.5 (1.3) 34.9 (1.0) 29.6 (1.2) 27.9 (1.2) 29.9 (1.2) 30.5 (1.0) 28.5 (1.0) 31.5 (1.2) 29.5 (1.0) 26.4 (1.0) 31.8 (1.6) 28.9 (1.3) 24.3 (0.9) 34.7 (1.1) 27.6 (1.8) 29.2 (0.6)
° Interaction effect, P ⬍ 0.05.
age dependent, as the number of neurons was lower in aged mice. Despite this neuropathology, no transgenerelated change of CO activity was observed in the Purkinje cells and the cerebellar cortex. The type II neurofilament-rich inclusions observed in NFH-LacZ Purkinje cells (38), suspected of causing cell degeneration, did not seem to induce a direct CO alteration in the cerebellar cortex. An electron microscopic study should determine more precisely the possible effect of type II inclusions on Purkinje cell metabolic activity and viability. CO activity is reported in values relative to tissue weight. Because the Purkinje cell measure is intracellular, the values reflect the activity of the remaining cells. The normal CO activity values in NFH-LacZ mice mean that the remaining Purkinje cells are as metabolically active as the nondegenerated tissue of agematched controls. The unchanged CO activity observed in the NFH-LacZ deep cerebellar nuclei confirms this hypothesis. By contrast, Lurcher and staggerer ataxic mice characterized by massive Purkinje cell loss, despite an equal preservation of cerebellar cortical metabolic activity, displayed higher CO activity than controls in the deep nuclei (8, 33), perhaps indicating that the Purkinje cell loss in NFH-LacZ mice is insufficient for causing this elevation (24).
CO activity was increased in the molecular and Purkinje cell layers of the cerebellum in both groups of aged mice. A similar phenomenon was observed among the remaining ectopic Purkinje cells of staggerer mutants (8). This increase may reflect a functional compensation to aging Purkinje cells or the initial stage of a degenerative process. Cerebellar-Related Regions The CO activity of NFH-LacZ mice was decreased in three cerebellar-related regions: (1) the parvicellular part of the red nucleus (12, 21, 29), (2) the superior colliculus (12), and (3) the lateral reticular nucleus (31), perhaps as a result of the changes in Purkinje cell volume. In addition to cerebellar projections, these regions are interconnected and establish similar connections to other regions. The lateral reticular nucleus, providing a mossy fiber pathway to the cerebellar cortex and deep nuclei, receives its main afferent input from the frontoparietal sensorimotor cortex, the superior colliculus, the contralateral red nucleus, and the spinal cord (31, 32). Similarly, the superior colliculus is connected with the spinal cord, the motor cortex, and the cerebellar deep nuclei (1, 25, 27). Concerning the red nucleus, physiological and anatomical studies in
526
STRAZIELLE ET AL.
TABLE 4 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in the Telencephalon of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control Region Cerebral cortex Motor° Eye field Lateral prefrontal Cingulate Orbital Prelimbic Retrosplenial Anterior insular Anterior parietal Posterior parietal Entorhinal # Auditory Visual Piriform Olfactory tubercle Nucleus accumbens Striatum Dorsal lateral Dorsal medial Lateral pallidum #
NFH
Young
Old
Young
Old
27.7 (1.6) 28.2 (1.5) 31.2 (0.8) 29.7 (1.5) 29.9 (0.8) 27.1 (1.2) 33.0 (1.8) 30.0 (0.8) 28.8 (1.0) 31.2 (1.0) 28.1 (1.1) 29.6 (0.8) 30.9 (1.3) 31.0 (0.9) 34.5 (1.1) 36.6 (1.8)
32.1 (1.2) 31.2 (1.0) 30.8 (1.1) 32.7 (0.9) 30.1 (0.9) 28.2 (1.2) 34.4 (1.2) 30.2 (0.7) 31.9 (0.9) 31.2 (0.9) 30.7 (1.4) 30.2 (1.1) 32.7 (1.1) 32.5 (1.3) 36.0 (2.1) 40.0 (1.8)
30.2 (0.9) 29.0 (0.8) 31.3 (1.8) 31.7 (1.2) 31.0 (2.1) 27.0 (1.3) 30.8 (1.3) 31.3 (1.2) 29.6 (1.3) 31.2 (1.4) 26.7 (0.8) 29.0 (0.8) 31.4 (0.8) 30.5 (0.9) 34.2 (2.5) 36.8 (2.3)
29.5 (0.8) 29.5 (0.5) 30.6 (1.0) 31.5 (0.6) 28.9 (1.1) 27.7 (1.1) 33.9 (0.8) 30.5 (1.0) 30.0 (1.0) 33.1 (1.1) 30.2 (1.2) 29.2 (1.5) 30.0 (0.8) 30.9 (0.7) 36.5 (1.6) 38.6 (1.6)
29.5 (1.1) 27.6 (1.1) 21.1 (1.2)
32.0 (1.3) 29.8 (1.5) 24.0 (1.0)
30.8 (1.1) 28.5 (1.1) 20.6 (1.7)
29.6 (1.3) 26.9 (1.0) 21.6 (1.1)
Age effect, P ⬍ 0.05; °interaction effect, P ⬍ 0.05.
the rat (15, 29, 37) indicate that magno- and parvicellular parts of the red nucleus are controlled by cerebral as well as cerebellar influences. While the magnocellular red nucleus receives a predominant input from the interpositus nucleus (7, 9), the parvicellular part receives a predominant input from the dentate nucleus and the sensorimotor cortex (7, 17, 21). The two subregions differ as well in terms of their efferent connections. The magnocellular red nucleus projects mostly to the spinal cord (7, 9), while the parvicellular part projects preferentially to the inferior olive (22, 34).
These different neuroanatomical circuitries are summarized in Fig. 3. One of the possible reasons for the CO alterations in these regions is that metabolic depression occurred as a secondary consequence of cerebellar dysfunction. The CO activity of cerebellar-related pathways was also altered in Lurcher (33) and staggerer (8) mutants, but in the opposite direction (increased activity), as a possible result of the higher CO activity found in the deep cerebellar nuclei of these mice. By contrast, hypometabolism is an indication of cell stress rather than a sign of a compensatory action,
TABLE 5 Cytochrome Oxidase Activity (Means and SEM in mol/min/g of Tissue) in White Matter Tracts of Young and Old NFH-LacZ and Control Mice (Means Differing Expressed in Bold Characters) Control
#
NFH
Region
Young
Old
Young
Old
Optic nerve Medial lemniscus Fornix Mammillothalamic Nigrostriatal Medial longitudinal Cerebellar vermis Cerebellar hemisphere # Medial forebrain bundle
13.6 (0.6) 25.2 (1.2) 8.7 (0.9) 13.5 (1.0) 24.6 (1.0) 14.4 (1.5) 7.9 (0.7) 7.1 (0.6) 21.4 (1.1)
14.6 (1.3) 25.6 (1.1) 10.0 (0.6) 15.3 (1.3) 26.7 (1.7) 15.8 (0.9) 9.1 (0.6) 9.0 (0.4) 24.1 (2.0)
12.5 (1.3) 25.6 (1.2) 9.9 (1.1) 12.7 (0.9) 24.3 (1.4) 13.4 (1.0) 8.7 (0.6) 7.6 (0.5) 21.0 (1.5)
12.9 (1.5) 24.2 (1.4) 8.9 (0.8) 13.0 (1.0) 23.4 (1.0) 13.4 (1.1) 9.4 (0.6) 8.7 (0.7) 21.6 (0.9)
Age effect, P ⬍ 0.05.
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
527
FIG. 1. CO labeling of the red nucleus analyzed in young control (a) and in old NFH-LacZ (b) mice. Note the lower CO intensity of labeling in parvicellular part of the transgenic mouse region. (Scale bar ⫽ 100 m.) mc, magnocellular part of the red nucleus; PAG, periaqueductal grey nucleus; pc, parvicellular part of the red nucleus; RED, red nucleus.
indicating, therefore, that the type II neurofilamentrich inclusions in Purkinje cells may cause dysfunctions in connected structures. It is interesting to note that type II inclusions were observed among 60% of neocortical neurons in NFH-LacZ mice (38). Although it is not known whether the motor cortex is reduced in volume, it may be hypothesized that the lower CO activity found in the brain stem may also be a direct consequence of cortical afferent dysfunction. In addition, the reduced axonal calibers of the motor neurons in the spinal cord (10), associated with decreased neuronal conduction, might contribute as well to the metabolic alteration of these three brain stem motor regions. Because of the existence of projections from the superior colliculus and the red nucleus to the lateral reticular nucleus (30; Fig. 3), it is possible that CO alterations in the latter are caused not only by transsynaptic changes secondary to cerebellar damage but also by transsynaptic changes secondary to the depressed metabolic activity in both the superior colliculus and red nucleus. The reduced CO activity in the superior colliculus may also be due to the decreased optic nerve caliber in these mice (10), but this hypothesis is not supported by the lack of CO activity changes in the optic nerve, an area with few mitochondria.
In conclusion, despite the fact that NFH is expressed in most neurons of the central and peripheral nervous system (20), CO alterations in NFH-LacZ mice were limited to cerebellar- or cortical-related regions in the brain stem, resulting in selective deficits of motor coordination and visuomotor guidance while swimming toward a visible goal (24). In particular, low CO activity in the lateral reticular nucleus was associated with poor motor performance and low CO activity in the superior colliculus was associated with poor visuomotor performance (24). These results emphasize the functional consequence of cerebellar-related structures in this form of transgene expression. The Accentuated Transgene Effect during Aging The interactions between the transgene factor and the aging factor were significant in six brain regions, including brain stem (dorsal motor nucleus of the vagus nerve, superior olive, and trapezoid nucleus) and forebrain (lateral dorsal nucleus of the thalamus, subiculum, and motor cortex). In these regions, aging changed CO activity in an opposite way in NFH-LacZ mice as opposed to controls. In particular, CO activity increased in aged controls relative to young controls in
528
STRAZIELLE ET AL.
FIG. 2. Detail of the cerebellar cortex in young (a) and in aged (b) NFH-LacZ mice. A more intense CO labeling is observed in the molecular and Purkinje cell layers of the aged mouse in comparison to the young mouse; the arrows show the Purkinje cells. (Scale bar ⫽ 100 m.) Gr, granular layer; mol, molecular layer; Pj, Purkinje cell layer.
most of these regions. This was not the case in NFHLacZ mice, as their activity was decreased during aging. Previous experiments have indicated regionally selective changes in neurofilament levels during aging (39, 43). The present study indicates cell dysfunction in selective regions of aged NFH-LacZ mice as a possible result of the perikaryal neurofilament accumulations. Aging Effects in Cerebellum and Related Pathways Irrespective of the presence or the absence of the transgene, aged mice had lower CO activity in the dentate nucleus of the cerebellum, the magnocellular part of the red nucleus, and the external cuneate nucleus. On the contrary, CO activity was increased in the molecular layer and Purkinje cells of the cerebellum, in the lateral cerebellar white matter, and in the entorhinal cortex. Because of inhibitory GABAergic connections between Purkinje cells and the deep cerebellar nuclei (28), it is possible that the increased CO activity in the cerebellar cortex is directly responsible for the reduced CO activity in the dentate nucleus. Decreased CO activity in the magnocellular red nucleus may be a precursor to cell atrophy (36), as attested by the significant cell reduction found in the present study, but it may also be explained by the diminished metabolic activity in the dentate, as these projections are excitatory (37). Another possible expla-
nation of the selective regional hypometabolism found in the magnocellular red nucleus and the external cuneate nucleus, site of primary muscle afferents (2, 35), is based on their connections with motoneurons in the spinal cord. It has been shown that aging causes losses of motoneurons and external cuneate proprioceptive neurons in mice as early as 12 months (35). Because the magnocellular neurons give rise to most of the rubrospinal tract (7, 9), the CO activity reduction in this region could result from the age-related loss of motoneurons. CONCLUSION
The present study demonstrated that the neurofilament maldistribution seen in NFH-LacZ mice affects the metabolic activity of selective neuronal populations of the central nervous system. The entrapment of cellular organelles such as mitochondria in the perikaryon may affect neuronal metabolic activity and possibly its viability as a function of aging. Mitochondrial abnormalities characterize several neurodegenerative diseases with neurofilament inclusions, particularly amyotrophic lateral sclerosis (3, 13, 26). The NFH-LacZ mice will provide an opportunity for studying the role played by neurofilament inclusions on the mitochondrial compartment of the cells in the pathogenesis of the disease.
REGIONAL BRAIN ACTIVITY IN NFH-LacZ TRANSGENIC MICE
529
FIG. 3. Functional circuitries between brain stem structures mainly involved in motor control. Cerebellar afferents reach the cerebellar cortex, while efferents leave the cerebellum via the deep nuclei. Boxes illustrating the different structures appear more lighted when a significantly lower CO activity was observed in the NFH-LacZ mice (transgene effects). Double-way connections between structures are drawn in bold characters.
ACKNOWLEDGMENTS
6.
This work was supported by a grant from the Association Franc¸ aise de l’Ataxie de Friedreich (AFAF) to C.S. and R.L., and from the Association Franc¸ aise contre les Myopathies (AFM) to J.E.. We thank P. Robert and P. Chiron for technical assistance (animal facility and genotyping) at Angers.
7.
8.
REFERENCES 1.
2.
3.
4.
5.
Antonetty, C. M., and K. E. Webster. 1975. The organization of the spinotectal projections: An experimental study in the rat. J. Comp. Neurol. 163: 449 – 466. Bolton, P. S., and D. J. Tracey. 1992. The medullary relay from neck receptors to somatosensory thalamus in the rat: A neuroanatomical study. Exp. Brain Res. 88: 473– 484. Borthwick, C. M., M. A. Johnson, P. G. Ince, P. J. Shaw, and D. M. Turnbull. 1999. Mitochondrial enzyme activity in amyotrophic lateral sclerosis: Implications for the role of mitochondria in neuronal cell death. Ann. Neurol. 46: 787–790. Carden, M. J., J. Q. Trojanowski, W. W. Schlaepter, and V. M.-Y. Lee. 1987. Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7: 3489 –3504. Collard, J.-F., F. Coˆ te´ , and J.-P. Julien. 1995. Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis. Nature 375: 61– 64.
9.
10.
11.
12.
13.
Coˆ te´ , F., J.-F. Collard, and J.-P. Julien. 1993. Progressive neuropathy in transgenic mice expressing the human neurofilament heavy gene: A mouse model of amyotrophic lateral sclerosis. Cell 73: 35– 46. Daniel, H., J. M. Billard, P. Angaut, and C. Batini. 1987. The interposito-rubrospinal system. Anatomical tracing of a motor control pathway in the rat. Neurosci. Res. 5: 87–112. Deiss, V., C. Strazielle, and R. Lalonde. 2000. Regional brain variations of cytochrome oxidase activity and motor co-ordination in staggerer mutant mice. Neuroscience 95: 903–911. Dekker, J. J. 1981. Anatomical evidence for direct fiber projections from the cerebellar nucleus interpositus to rubrospinal neurons. A quantitative EM study in the rat combining anterograde and retrograde intra-axonal tracing methods. Brain Res. 205: 229 –244. Eyer, J., and A. Peterson. 1994. Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament--galactosidase fusion protein. Neuron 12: 389 – 405. Eyer, J., D. W. Cleveland, P. C. Wong, and A. Peterson. 1998. Pathogenesis of two axonopathies does not require axonal neurofilaments. Nature 391: 584 –587. Faull, R. L. M., and J. B. Carman. 1978. The cerebellofugal projections in the brachium conjunctivum of the rat. I. The contralateral ascending pathway. J. Comp. Neurol. 178: 495–518. Forno, L. S. 1986. The Lewy body in Parkinson’s disease. Adv. Neurol. 45: 35– 43.
530 14. 15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
STRAZIELLE ET AL. Franklin, K. B. J., and G. Paxinos. 1997. The Mouse Brain in Stereotaxic Coordinates. Academic Press, New York. Giuffrida, R., G. Li Volsi, and V. Perciavalle. 1988. Influences of cerebral cortex and cerebellum on the red nucleus of the rat. Behav. Brain Res. 28: 109 –111. Gonzalez-Lima, F., and D. Jones. 1994. Quantitative mapping of cytochrome oxidase activity in the central auditory system of the gerbil: A study with calibrated activity standards and metalintensified histochemistry. Brain Res. 660: 34 –39. Gwyn, D. G., and P. A. Flumerfelt. 1974. A comparison of the distribution of cortical and cerebellar afferents in the red nucleus of the rat. Brain Res. 69: 130 –135. Harley, C. A., and C. H. Bielajew. 1992. A comparison of glycogen phosphorylase a and cytochrome oxidase histochemical staining in rat brain. J. Comp. Neurol. 322: 377–389. Hess, H. H., and A. Pope. 1953. Ultramicrospectrophotometric determination of cytochrome oxidase for quantitative histochemistry. J. Biol. Chem. 204: 295–306. Julien, J.-P., D. Meijer, J. Hurst, and F. Grosveld. 1986. Cloning and developmental expression of the murine neurofilament gene family. Mol. Brain Res. 1: 243–250. Kennedy, P. R., A. R. Gibson, and J. C. Houk. 1986. Functional and anatomic differentiation between parvicellular and magnocellular regions of red nucleus in the monkey. Brain Res. 364: 124 –136. Kennedy, P. R. 1990. Corticospinal, rubrospinal and rubroolivary projections: A unifying hypothesis. Trends Neurosci. 13: 474 – 479. Kre´ marik, P., C. Strazielle, and R. Lalonde. 1998. Regional brain variations of cytochrome oxidase activity and motor coordination in hot-foot mutant mice. Eur. J. Neurosci. 10: 2802– 2809. Lalonde, R., M. Dubois, C. Strazielle, and J. Eyer. 1999. Motor coordination and spatial orientation are affected by neurofilament maldistribution: Correlations with regional brain activity of cytochrome oxidase. Exp. Brain Res. 126: 223–234. Lee, H. S., R. J. Kosinski, and G. A. Mihailoff. 1989. Collateral branches of cerebellopontine axons reach the thalamus, superior colliculus or inferior olive: A double fluorescence and combined fluorescence-horseradish peroxidase study in the rat. Neuroscience 28: 725–735. Murayama, S., Y. Ookawa, H. Mori, I. Nakano, Y. Ihara, S. Kuzuhara, and M. Tomonaga. 1989. Immunocytochemical and ultrastructural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis. Acta Neuropathol. 78: 143–152. Myashita E., E. Keller, and H. Asuma. 1994. Input-output organization of the rat vibrissal motor cortex. Exp. Brain Res. 99: 223–232.
28. 29.
30.
31.
32.
33.
34.
35. 36. 37.
38.
39.
40.
41.
42.
43.
Oertel, W. H. 1993. Neurotransmitters in the cerebellum: Scientific aspects and clinical relevance. Adv. Neurol. 61: 33–75. Perciavalle, V., S. Berretta, and R. Raffaele. 1989. Projections from the intracerebellar nuclei to the ventral midbrain tegmentum in the rat. Neuroscience 29: 109 –119. Qvist, H., and E. Dietrichs. 1985. The projection from the superior colliculus to the lateral reticular nucleus in the cat as studied with retrograde transport of WGA-HRP. Anat. Embryol. 173: 269 –274. Rajakumar, N., A. W. Hrycyshyn, and B. A. Flumerfelt. 1992. Afferent organization of the lateral reticular nucleus in the rat: An anterograde tracing study. Anat. Embryol. 185: 25–37. Shokunbi, M. T., A. W. Hrycyshyn, and B. A. Flumerfelt. 1986. A horseradish peroxidase study of the rubral and cortical afferents to the lateral reticular nucleus in the rat. J. Comp. Neurol. 248: 441– 454. Strazielle, C., P. Kre´ marik, J.-F. Ghersi-Egea, and R. Lalonde. 1998. Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice. Exp. Brain Res. 121: 35– 45. Strominger, N. L., T. C. Truscott, R. A. Miller, and G. J. Royce. 1979. An autoradiographic study of the rubro-olivary tract in the rhesus monkey. J. Comp. Neurol. 183: 33– 46. Sturrock, R. R. 1989. Loss of neurons from the external cuneate of the ageing mouse brain. J. Anat. 163: 135–141. Sturrock, R. R. 1990. Age related changes in neuron number in the mouse red nucleus. J. Hirnforsch. 31: 399 – 403. Toyama, K., N. Tsukuhara, and M. Udo. 1967. Nature of cerebellar influences upon the red nucleus neurones. Exp. Brain Res. 4: 292–309. Tu, P.-H., K. A. Robinson, F. de Snoo, J. Eyer, A. Peterson, V. M.-Y. Lee, and J. Q. Trojanowski. 1997. Selective degeneration of Purkinje cells with Lewy body-like inclusions in aged NFHLacZ transgenic mice. J. Neurosci. 17: 1064 –1074. Van Der Zee, E. A., P. A. Naber, and J. F. Disterhoft. 1997. Age-dependent changes in the immunoreactivity for neurofilaments in rabbit hippocampus. Neuroscience 79: 103–116. Wong-Riley, M. T. T. 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171: 11–28. Wong-Riley, M. T. T. 1989. Cytochrome oxidase: An endogenous metabolic marker for neuronal activity. Trends Neurosci. 12: 94 –101. Xu Z., D. L.-Y. Dong, and D. W. Cleveland. 1994. Neuronal intermediate filaments: New progress on an old subject. Curr. Opin. Neurobiol. 4: 655– 661. Zhang, J.-H., S. Sampogna, F. R. Morales, and M. H. Chase. 1997. Age-related alterations in immunoreactivity of the midsized neurofilament subunit in the brainstem reticular formation of the cat. Brain Res. 769: 196 –200.