TracMouse: A computer aided movement analysis script for the mouse inverted horizontal grid test.

In rodents, detection and quantification of motor impairments is difficult. The traction test (inverted grid with mice clinging to the underside) currently has no objective rating system. We here developed and validated the semi-automatic MATLAB script TracMouse for unbiased detection of video-recorded movement patterns. High precision videos were analyzed by: (i) principal identification of anatomical paw details frame-by-frame by an experimentally blinded rater; (ii) automatic retrieval of proxies by TracMouse for individual paws. The basic states of Hold and Step were discriminated as duration and frequency, and these principle parameters were converted into static and dynamic endpoints and their discriminating power assessed in a dopaminergic lesion model. Relative to hind paws, forepaws performed ~4 times more steps, they were ~20% longer, and Hold duration was ~5 times shorter in normal C57Bl/6 mice. Thus, forepaw steps were classified as exploratory, hind paw movement as locomotive. Multiple novel features pertaining to paw sequence, step lengths and exploratory touches were accessible through TracMouse and revealed subtle Parkinsonian phenotypes. Novel proxies using TracMouse revealed previously unidentified features of movement and may aid the understanding of (i) brain circuits related to motor planning and execution, and (ii) phenotype detection in experimental models of movement disorders.

Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer's disease and frontotemporal lobar degeneration.

A poorly understood feature of the tauopathies is their very different clinical presentations. The frontotemporal lobar degeneration (FTLD) spectrum is dominated by motor and emotional/psychiatric abnormalities, whereas cognitive and memory deficits are prominent in the early stages of Alzheimer's disease (AD). We report two novel mouse models overexpressing different human tau protein constructs. One is a full-length tau carrying a double mutation [P301S/G335D; line 66 (L66)] and the second is a truncated 3-repeat tau fragment which constitutes the bulk of the PHF core in AD corresponding to residues 296-390 fused with a signal sequence targeting it to the endoplasmic reticulum membrane (line 1; L1). L66 has abundant tau pathology widely distributed throughout the brain, with particularly high counts of affected neurons in hippocampus and entorhinal cortex. The pathology is neuroanatomically static and declines with age. Behaviourally, the model is devoid of a higher cognitive phenotype but presents with sensorimotor impairments and motor learning phenotypes. L1 displays a much weaker histopathological phenotype, but shows evidence of neuroanatomical spread and amplification with age that resembles the Braak staging of AD. Behaviourally, the model has minimal motor deficits but shows severe cognitive impairments affecting particularly the rodent equivalent of episodic memory which progresses with advancing age. In both models, tau aggregation can be dissociated from abnormal phosphorylation. The two models make possible the demonstration of two distinct but nevertheless convergent pathways of tau molecular pathogenesis. L1 appears to be useful for modelling the cognitive impairment of AD, whereas L66 appears to be more useful for modelling the motor features of the FTLD spectrum. Differences in clinical presentation of AD-like and FTLD syndromes are therefore likely to be inherent to the respective underlying tauopathy, and are not dependent on presence or absence of concomitant APP pathology.

Nerve growth factor differentially affects spatial and recognition memory in aged rats.

In rats, object discrimination depends on the integrity of the cholinergic system, thus it could be expected that nerve growth factor (NGF) can improve the behavior in aged subjects. The interactive effect of age and cholinergic improvement was assessed behaviorally in young and aged rats. Animals were injected by infusion of NGF into the lateral ventricles and they were tested in two behavioral tasks: an object-location and an object-recognition task. Spatial and recognition memory were assessed in an open field containing five different objects. Rats were submitted to six consecutive sessions. Both age-groups showed comparable habituation of exploratory response in Session 1-4. Discrimination index (DI) was calculated to assess responses to spatial change in Session 5 and object change in Session 6. Control young and aged rats were able to discriminate between familiar and novel object, however DI was lower in aged rats. Treatment with NGF induced decline of object discrimination in both age-groups. Different results were obtained in spatial displacement test. NGF was able to improve spatial memory in aged rats, but had no effect in young controls. These data confer on NGF potential role in improving spatial but not episodic memory in aged rats.

Compartmental protein expression of Tau, GSK-3beta and TrkA in cholinergic neurons of aged rats.

During aging basal forebrain cholinergic neurons (BFCNs) degenerate, and we hypothesize this to be the result of a degeneration of the cytoskeleton. As a corollary, retrograde transport of the complex of nerve growth factor (NGF) and its activated receptor phospho-TrkA (P-TrkA) is impaired. Using immunocytochemistry, we here compare young and aged rat brains in their subcellular localization of NGF and P-TrkA in relation to the compartmentalization of phosphorylation-dependent tau protein isoforms. Despite lower P-TrkA immunoreactivity in cortex and hippocampus of aged rats, NGF immunoreactivity was not altered in these areas, but was significantly lower in aged basal forebrain. In young animals, expression of tau isoforms and glycogen synthase kinase-3beta (GSK-3beta) was restricted to neuritic structures in cortex, hippocampus, and basal forebrain. In contrast, tau and GSK-3beta labeling was confined to cell bodies in aged rats. Since a somatic localization of phospho-tau is indicative of cytoskeletal breakdown, we suggest this to be the mechanism the breakdown of trophic support in aging BFCNs.

Does senile impairment of cholinergic system in rats concern only disturbances in cholinergic phenotype or the progressive degeneration of neuronal cell bodies?

The trophic effect of continuous intraventricular infusion of nerve growth factor (NGF) on morphology of the basal forebrain (BF) cholinergic neurons was tested in 4- and 28-month-old male Wistar rats. All studies were conducted using behaviorally uncharacterized animals from the same breeding colony. Immunohistochemical procedure for choline acetyltransferase (ChAT) and p75NTR receptor has been applied to identify cholinergic cells in the structures of basal forebrain (BF). Using a quantitative image analyzer, morphometric and densitometric parameters of ChAT- and p75NTR-positive cells were measured immediately after cessation of NGF infusion. In 28-month-old non-treated rats the number of intensively ChAT-positive cells in all forebrain structures was reduced by 50-70% as compared with young animals. The remaining ChAT-positive cells appeared shrunken and the neuropil staining was NTR markedly reduced. In contrast, the same neurons when stained for p75 were numerous and distinctly visible with perfect morphology. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neuronal cell bodies. NGF restored the number of intensely stained ChAT-positive cells to about 90% of that for young controls and caused a significant increase in size of those cells in 28-month-old rats as compared with the control, age-matched group. NGF did not influence the morphology of p75NTR-positive neurons, which were well labeled, irrespective of treatment and age of the rats. In 4-month-old rats, NGF infusion decreased the intensity of both ChAT and p75NTR immunostaining. These data provide some evidence for preservation of BF cholinergic neurons from atrophy during aging and indicate that senile impairment of the cholinergic system in rats concerns decrease in ChAT-protein expression rather than an acute degeneration of neuronal cell bodies. Treatment with NGF resulted in restoration of cholinergic phenotype in the BF neurons of aged rats. However, the present study also rises issue of possible detrimental effects of NGF in young normal animals.

[The cerebral cortex: structure, interrelations of neurons, functional columnar organization]. / Kora mózgowa: struktura, polaczenia miedzykomórkowe, funkcjonalna organizacja kolumnowa.

The cerebral cortex develops from a portion of the telencephalic vesicle. Cells originating from the germinal zone surrounding the lumen migrate peripherally to form the cortical mantle. During the fetal life, cortical neurons begin to form six horizontal layers. Cells formed at the same time migrate to the same cortical layer, cells migrating later pass through deep layers to form more superficial laminae. A six layer cellular organization is characteristic of the entire neopallium, which is referred to as the neocortex. The paleopallium--olfactory cortex--and the archipallium--hippocampal formation and dentate gyrus--have three basic layers and collectively constitute the allocortex. Through the course of evolution, the cerebral cortex has increased enormously in surface area but not in thickness, a trend that can be seen even in the relatively short period of primate evolution. The human cortex is only about 15% thicker than that of the macaque monkey but is at least 10 times greater in area. To understand how this complex structure functions we need to examine its unique internal cellular arrangement.

Subchronic intraventricular infusion of quinolinic acid produces working memory impairment--a model of progressive excitotoxicity.

It has been proposed by Yamada et al. [Neurosci. Lett. 118: 128-131 (1990); J. Pharmacobiodyn. 14: 351-355 (1991)] that subchronic i.c.v. infusion of the NMDA receptor agonist quinolinic acid may serve as a model for some aspects of neurodegenerative dementia. In the present study, quinolinic acid (9 mM) was infused i.c.v. by ALZET osmotic minipumps for 2 weeks. This treatment produced a short-term working memory deficit in the T-maze (alternation) but no change in reversal learning in the same test. The working memory deficit in the T-maze was progressive i.e. seen after 14, but not 3 days of infusion and persisted for at least for 3 weeks after the termination of the infusion. Histological examination revealed a modest decrease in the number of cells in the nucleus basalis magnocellularis but not in the striatum, entorhinal cortex, or hippocampus. However, in most of the structures studied, morphological changes such as swollen somata and irregular shape were observed indicative of alterations in neuronal function. Autoradiography in the hippocampus revealed a decrease in [3H]hemicholinium and [3H]quinuclidinyl benzilate (QNB) binding to choline uptake sites and muscarinic receptors respectively. Surprisingly no change was observed in [3H]MK-801 binding to NMDA receptor channels in the hippocampus and cortex. The subchronic infusion of quinolinic acid may serve as a model of progressive deterioration of cognitive functions.

Postsynaptic and extrasynaptic localization of kappa-opioid receptor in selected brain areas of young rat and chick using an anti-receptor monoclonal antibody.

kappa-opioid receptors were visualized by light and electron microscopical immunohistochemistry in young rat and chick brains, using a monoclonal antibody KA8 (IgG1, kappa) raised against a kappa-opioid receptor preparation from frog brain, which recognizes selectively the kappa-type receptor with preference for the kappa-2 subtype. The most pronounced kappa-opioid receptor-like immunoreactivity was observed in the hypothalamic nuclei of the rat brain and in the chick optic tectum, in regions where the functional significance of kappa-opioid receptors is well documented. Both neurons and glia were stained, the former on both somata and dendrites. At the ultrastructural level, the receptor-like immunoreactivity was similar in both species. Immunoprecipitate decorated the inner surface of the plasma membrane of glial cells, neuronal somata and dendrites, in a discontinuous arrangement. In the cytoplasm, labelling was associated with ribosomes, polyribosomes and rough endoplasmic reticulum membranes but not with Golgi cisternae. In the neuropil, the immunoprecipitate was observed along the dendritic microtubules and was also associated with postsynaptic sites. Nuclei and axons were devoid of label and immunoreactivity was never visible presynaptically. Our findings indicate that the antibody used in the present study marks various forms of the kappa-opioid receptor protein including those synthesised in ribosomes, transported along dendritic microtubules and incorporated into postsynaptic and non-synaptic membranes. The antibody also recognizes glial opioid receptors. The observed subcellular distribution appears to be conserved in phylogenetically distant species.

The differences in learning abilities between spontaneously hypertensive (SHR) and Wistar normotensive rats are cue dependent.

We examined the performance of spontaneously hypertensive (SHR) and Wistar normotensive (NT) rats in acquisition, retention after a 2-month interval, and reversal learning in two tasks: simultaneous brightness discrimination (Experiment I) and conditional discrimination of directional locomotor responses (Experiment II). In both tasks food reinforcement was used. In Experiment I both SHR and NT groups comprised younger (3-month-old) and older (10-month-old) rats. In each experimental stage SHRs of both age groups mastered the task earlier and made fewer errors than the respective NT groups. Reversal learning took longer than acquisition of discrimination in both age groups of NT rats. Conversely, reversal learning was an easier task for SHR. In Experiment II only younger rats were used. The forced turn at the start in the modified T-maze was utilized as the cue to guide performance at the choice point of the maze. In acquisition and retention, rats were trained to select at the choice point the arm in the same direction as in the forced turn; in the reversal, opposite contingencies were applied. At all stages the choice accuracy of SHR was the same as that in NT rats. The contrasting findings of Experiment I and Experiment II indicate that SHR learned better than NT when exteroceptive visual stimuli were used, but performed at the same level as NT rats in the task where interoceptive kinesthetic cues were relevant. We suggest that SHR pay more attention to visual stimuli than NT rats.

Visual discrimination learning in spontaneously hypertensive (SHR) and Wistar normotensive rats: computerized analysis of choice strategies.

Spontaneously hypertensive rats (SHR) were found to learn a visual discrimination paradigm better than Wistar normotensive rats (NT). The present analysis of choice strategies used by rats was performed to detect possible correlates between patterns of responding and duration of learning. The following strategies were included into the analysis: perseveration and alternation of choice (response-set hypotheses), win-stay lose-shift and win-shift lose-stay (prediction hypotheses). A computer programme called "The Thinking Rat" was written to analyse rat strategies during the learning. The analysis was applied for two irrelevant dimensions of stimuli, i.e. position and orientation, and for colour as a relevant cue. SHR solved the task using different strategies than the NT rats. All subjects began by responding to position as a cue, but SHR gave up this strategy more quickly than NT rats did and responded earlier to colour. NT rats showed a strong preference to persevere in their choices with respect to stimulus position, whereas SHR used more frequently the prediction hypotheses with respect to colour. The analysis of performance and the analysis of choice strategies-could indicate that the discrepancy in learning between SHR and NT rats may be caused by differences in attention.

Regional and laminar variations in acetylcholinesterase activity within the frontal cortex of the dog.

Two different histochemical methods were applied to analyse acetylcholinesterase (AChE) activity within the frontal lobe cortex (FC) of the dog. Both staining methods revealed AChE reactivity in neuronal cell bodies and fibres. AChE-positive neuronal perikarya varied in size, shape, character and intensity of staining. Both pyramidal and non-pyramidal AChE-rich neurons were found. The pyramidal neurons predominated in layers III and V of the dog FC. The non-pyramidal cells were present in deep cortical layers and white matter. Labelled cells were distributed in a consistent pattern across regions of the dog frontal lobe. AChE reactivity in fibres showed, in general, a characteristic bilaminar appearance due to the more intense staining in cortical layers I and V. However, in contrast to the cellular labelling, differences in the laminar distribution of AChE-rich fibre bands distinguished three subregions of the FC: (1) rostral and middle prefrontal and anterior premotor areas, where AChE was distributed in a bilaminar pattern with two bands of similar, medium-intensive staining overlying layers I and V; (2) dorso-caudal primary and secondary motor areas distinguished by much lighter staining of the deep band of AChE activity in layer V; and (3) ventro-caudal subcallosal region in which the bilaminar pattern of extremely dark labelling in layers I and V was augmented by a third band of strong AChE activity in layer VI. These findings show that differences in the pattern of AChE activity parallel some of the cytoarchitectonic zones of the FC previously described in this laboratory (Rajkowska and Kosmal, 1988).

Some evidence of enhanced alimentary motivation in spontaneously hypertensive rats (SHR).

The consumption of a wet mash of biscuits or pellets, outside the home cage, was determined in adult spontaneously hypertensive (SHR) and normotensive (NT) Wistar rats. Rats were deprived of food for 24 h or satiated. Regardless of the testing conditions, SHR showed a higher food interest than NT rats, which was reflected in a shorter latency to start eating, a longer time of eating to the first interval as well as to the 30 sec interval which terminated the trial, and a larger amount of food consumed during the test trial. The speed of eating was greater in SHR. In the competition for food SHR won almost all encounters with NT rats. The mean time of eating by SHR increased over the consecutive encounters up to 90%. These findings indicate that alimentary motivation of SHR was higher than that of NT rats. The results are discussed in terms of hyperreactivity of SHR.

Increase in body weight of spontaneously hypertensive rats (SHR) under prolonged behavioral stimulation.

We noticed that during 44 weeks of exposure to a number of behavioral tests (such as assessments of emotionality, locomotion, short term memory and visual discrimination learning) the body weight of spontaneously hypertensive rats (SHR) increased above that of normotensive controls (NT). To determine whether this difference was related to "behavioral stimulation" resulting from different testing situations and accompanying handling, body weights were recorded in other SHR and NT groups not subjected to any experiment (unstimulated). Stimulated SHR were significantly heavier than rats from other subgroups (unstimulated SHR as well as stimulated and unstimulated NT rats) which did not differ from one another. The first significant difference appeared after 7 weeks of behavioral training (18th week of life). Several weeks later, and continuing for the duration of study, stimulated SHR were approximately 12% heavier than the other 3 groups. No effect of behavioral testing on blood pressure was seen in either strain.