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.

Spatial learning impairments in PLB1Triple knock-in Alzheimer mice are task-specific and age-dependent.

We recently generated an advanced mouse model of Alzheimer's disease (AD) by targeted knock-in of single-copy mutated human amyloid precursor-protein (APP) and tau genes, crossed with a non-symptomatic presenilin (PS1A246E) over-expressing mouse line. These PLB1Triple mice presented with age-dependent and AD-relevant phenotypes. Homozygous PLB1Triple mice aged 4-12 months were assessed here in a battery of spatial learning tasks: Exp.1 radial-arm water maze (spatial reference and working memory) Exp.2 open-field water maze (spatial reference memory); Exp.3 home cage observation system with spatial learning (IntelliCage); Exp.4 spontaneous object recognition (SOR; novel object and spatial object shift). A separate test with high-expression transgenic APP mice matching the design of experiment 1 was also performed. Spatial deficits in PLB1Triple mice were confirmed at 12, but not 4 months in both water maze tasks. PSAPP mice, by contrast, presented with severe yet non-progressive spatial learning deficits already at 4 months. During tests of spatial learning in SOR and IntelliCage, PLB1Triple mice neither acquired the location of the water-rewarded corner, nor recognize novel or spatially shifted objects at 4 months, indicating these protocols to be more sensitive than the water maze. Collectively and in line with AD symptomatology, PLB1Triple mice present with a graded and progressive age-dependent loss of spatial memory that can be revealed by the use of a battery of tasks. With the emergence of subtle deficits progressively increasing in severity, PLB1Triple mice may offer a more patho-physiologically relevant model of dementia than aggressive expression models.

Long-term use of HU210 adversely affects spermatogenesis in rats by modulating the endocannabinoid system.

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of Δ9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm's endocannabinoid system. Long-term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users. HU210, a selective agonist for CB1 and CB2 cannabinoid receptors impairs spermatogenesis and sperm motility and deregulates the endocannabinoid system.

Acetylcholine: future research and perspectives.

Ever since the initial description of chemical transmission in the early part of the 20th century and the identification of acetylcholine (ACh) as the first such transmitter, interests grew to define the multiple facets of its functions. This multitude is only partially covered here, but even in the areas preselected for this special issue, research on the cholinergic system is still thriving. Notwithstanding an impressive amount of knowledge that has been accumulated, partly triggered by the cholinergic hypothesis of Alzheimer's disease (AD [1]), the different reviews in this issue not only summarise our current state of the art, they also highlight that this field has still large potential for future development. Taken from these reviews, we here pinpoint several topics fit for future attention.

Scopolamine-induced deficits in social memory in mice: reversal by donepezil.

Deficits in social behaviour is a characteristic of numerous mental disorders including autism, schizophrenia, depression and Alzheimer's disease. For the assessment of pharmacological and genetic experimental disease models, conventional social interaction tasks bear the uncertainty that any drug-induced abnormality of the investigator may feed back to the drug-free companion modifying its reactions. A considerable technical improvement was recently reported by Moy et al. [Moy SS, Nadler JJ, Perez A, Barbaro RP, Johns JM, Magnuson T, et al. Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behaviours in mice. Genes Brain Behav 2004;3:287-302] in which the drug free partner is confined to a small cage and social contacts of the investigator are recorded uncontaminated of any social reactions of the stranger. Using this novel behavioural paradigm, we here show in C57Bl/6 female mice that sociability (social interaction with a stranger mouse) is not impaired after administration of the anxiolytic diazepam (0.1-1 mg/kg) or the muscarinic antagonist scopolamine hydrobromide (0.1-1 mg/kg). However, social memory tested after a short time interval was impaired by both drugs in a dose-dependent manner (diazepam: > or = 0.5mg/kg; scopolamine: > or = 0.3mg/kg). The scopolamine-induced short-term memory deficit was reversed to normal by the choline esterase inhibitor donepezil (1 mg/kg). Given this dependence of social recognition on the cholinergic system, combined with the clinical observation of reduced social contacts in dementia patients, sociability may offer a novel endpoint biomarker with translational value in experimental models of cognitive dysfunction.

The synthetic cannabinoid HU210 induces spatial memory deficits and suppresses hippocampal firing rate in rats.

BACKGROUND AND PURPOSE: Previous work implied that the hippocampal cannabinoid system was particularly important in some forms of learning, but direct evidence for this hypothesis is scarce. We therefore assessed the effects of the synthetic cannabinoid HU210 on memory and hippocampal activity. EXPERIMENTAL APPROACH: HU210 (100 microg kg(-1)) was administered intraperitoneally to rats under three experimental conditions. One group of animals were pre-trained in spatial working memory using a delayed-matching-to-position task and effects of HU210 were assessed in a within-subject design. In another, rats were injected before acquisition learning of a spatial reference memory task with constant platform location. Finally, a separate group of animals was implanted with electrode bundles in CA1 and CA3 and single unit responses were isolated, before and after HU210 treatment. KEY RESULTS: HU210 treatment had no effect on working or short-term memory. Relative to its control Tween 80, deficits in acquisition of a reference memory version of the water maze were obtained, along with drug-related effects on anxiety, motor activity and spatial learning. Deficits were not reversed by the CB(1) receptor antagonists SR141716A (3 mg kg(-1)) or AM281 (1.5 mg kg(-1)). Single unit recordings from principal neurons in hippocampal CA3 and CA1 confirmed HU210-induced attenuation of the overall firing activity lowering both the number of complex spikes fired and the occurrence of bursts. CONCLUSIONS AND IMPLICATIONS: These data provide the first direct evidence that the underlying mechanism for the spatial memory deficits induced by HU210 in rats is the accompanying abnormality in hippocampal cell firing.

Dissociation of cholinergic function in spatial and procedural learning in rats.

The cholinergic system has long been known for its role in acquisition and retention of new information. Scopolamine, a muscarinic acetylcholine receptor antagonist impairs multiple memory systems, and this has promoted the notion that drug-induced side effects are responsible for diminished task execution rather than selective impairments on learning and memory per se. Here, we revisit this issue with the aim to dissociate the effects of scopolamine (0.2-1.0 mg/kg) on spatial learning in the water maze. Experiments 1 and 2 showed that acquisition of a reference memory paradigm with constant platform location is compromised by scopolamine independent of whether the animals are pre-trained or not. Deficits were paralleled by drug induced side-effects on sensorimotor parameters. Experiment 3 explored the role of muscarinic receptors in acquisition of an episodic-like spatial delayed matching to position (DMTP) protocol, and scopolamine still caused a learning deficit and side-effects on sensorimotor performance. Rats extensively pre-trained in the DMTP protocol with 30 s and 1 h delays over several months in experiment 4 and tested in a within-subject design under saline and scopolamine had no sensorimotor deficits, but spatial working memory remained compromised. Experiment 5 used the rising Atlantis platform in the DMTP paradigm. Intricate analysis of the amount of dwelling and its location revealed a clear deficit in spatial working memory induced by scopolamine, but there was no effect on sensorimotor or procedural task demands. Apart from the well-known contribution to sensorimotor and procedural learning, our findings provide compelling evidence for an important role of muscarinic acetylcholine receptor signaling in spatial episodic-like memory.

Global changes in the hippocampal proteome following exposure to an enriched environment.

Exposure to an enriched environment promotes neurochemical, structural and neurophysiological changes in the brain and is associated with enhanced synaptic plasticity and improved hippocampal-dependent learning. Using a global proteomics-based approach we have now been able to reveal the altered expression of a diverse range of hippocampal proteins following exposure to an enriched environment. Male Hooded Lister rats (8 weeks) were subjected to a 6-week regimen in which they were housed in either non-enriched (open field) or enriched conditions (toys, wheels etc.). Whole protein extracts from stratum pyramidale and stratum radiatum of area CA1 were then isolated and subjected to differential gel electrophoresis [McNair K, Davies CH, Cobb SR (2006) Plasticity-related regulation of the hippocampal proteome. Eur J Neurosci 23(2):575-580]. Of the 2469 resolvable protein spots detected in this study, 42 spots (1.7% of the detectable proteome) derived from predominantly somatic fractions and 32 proteins spots from dendritic fractions (1.3% of detectable proteome) were significantly altered in abundance following exposure to an enriched environment (somatic: 14 increased/28 decreased abundance, range -1.5 to +1.4-fold change; dendritic: 16 increased, 16 decreased abundance, range -1.6 to +3.0-fold change). Following in-gel tryptic digestion and Maldi-Tof/Q-star mass spectrometry, database searching revealed the identity of 50 protein spots displaying environmental enrichment-related modulation of expression. Identified proteins belonged to a variety of functional classes with gene ontology analysis revealing the majority (>70%) of regulated proteins to be part of the energy metabolism, cytoplasmic organization/biogenesis and signal transduction processes.

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.

Long-term homocysteine exposure induces alterations in spatial learning, hippocampal signalling and synaptic plasticity.

Abnormally high levels of homocysteine (HCY) have been linked to neurodegenerative diseases, but it remains unclear whether this is the cause or effect of degenerative processes. Here, we investigated the effects of prolonged HCY exposure on cognitive abilities and physiological parameters by injecting rats daily with either 20 or 200 mg/kg HCY over a period of up to 14 weeks. Notwithstanding a significant weight reduction in the 200 mg HCY group, HCY-exposed animals did not show a behavioural deficit when tested repeatedly (in weeks 1, 3, 5, 7 and 13) in a reference memory version of the water maze. Unexpectedly, some improvement in repeated reversal learning was observed in HCY exposed animals compared to controls. Pre-treatment with HCY for 3 weeks before water maze training did not uncover any cognitive alterations. Increased plasma concentrations of HCY were revealed only for the 200 mg HCY group after 14 weeks of injections, but no evidence for DNA damage was obtained. Immunocytochemically, HCY was detected in the brain after 14 weeks of treatment (both 20 and 200 mg/kg), but not after 5 weeks. Bidirectional changes in basic synaptic transmission and long-term potentiation of hippocampal CA1 pyramidal cells were observed at 5, 7 and 14 weeks in both HCY groups, indicative of complex, multifactorial time- and concentration-dependent changes. Overall, it is concluded that healthy adult rats are able to cope with continuous exposure to HCY. While HCY affects growth and neuronal excitability, this does not precipitate into an immediate impairment of cognitive function.

Cannabinoid function in learning, memory and plasticity.

Marijuana and its psychoactive constituents induce a multitude of effects on brain function. These include deficits in memory formation, but care needs to be exercised since many human studies are flawed by multiple drug abuse, small sample sizes, sample selection and sensitivity of psychological tests for subtle differences. The most robust finding with respect to memory is a deficit in working and short-term memory. This requires intact hippocampus and prefrontal cortex, two brain regions richly expressing CB1 receptors. Animal studies, which enable a more controlled drug regime and more constant behavioural testing, have confirmed human results and suggest, with respect to hippocampus, that exogenous cannabinoid treatment selectively affects encoding processes. This may be different in other brain areas, for instance the amygdala, where a predominant involvement in memory consolidation and forgetting has been firmly established. While cannabinoid receptor agonists impair memory formation, antagonists reverse these deficits or act as memory enhancers. These results are in good agreement with data obtained from electrophysiological recordings, which reveal reduction in neural plasticity following cannabinoid treatment, and increased plasticity following antagonist exposure. The mixed receptor properties of the pharmacological tool, however, make it difficult to define the exact role of any CB1 receptor population in memory processes with any certainty. This makes it all the more important that behavioural studies use selective administration of drugs to specific brain areas, rather than global administration to whole animals. The emerging role of the endogenous cannabinoid system in the hippocampus may be to facilitate the induction of long-term potentiation/the encoding of information. Administration of exogenous selective CB1 agonists may therefore disrupt hippocampus-dependent learning and memory by 'increasing the noise', rather than 'decreasing the signal' at potentiated inputs.

Reversible hippocampal inactivation partially dissociates how and where to search in the water maze.

To assess the interaction between spatial and procedural memory systems, the authors developed a learning protocol in the water maze using a rising "Atlantis" platform that requires rats to learn to swim to a specific location and, once there, to learn a "dwelling" response to raise the platform. In this protocol, the effects of temporal inactivation of the dorsal hippocampus with the AMPA/kainate receptor antagonist LY326325 on different memory phases were investigated. Hippocampal inactivation impaired acquisition of the searching task, mainly because of disruption of procedural learning. Inactivation also impeded consolidation and retrieval of spatial information, while leaving expression of dwelling responses intact. These findings challenge the idea of a sharp demarcation between spatial and procedural learning with respect to hippocampal involvement.

Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory.

The hypothesis that synaptic plasticity is a critical component of the neural mechanisms underlying learning and memory is now widely accepted. In this article, we begin by outlining four criteria for evaluating the 'synaptic plasticity and memory (SPM)' hypothesis. We then attempt to lay the foundations for a specific neurobiological theory of hippocampal (HPC) function in which activity-dependent synaptic plasticity, such as long-term potentiation (LTP), plays a key part in the forms of memory mediated by this brain structure. HPC memory can, like other forms of memory, be divided into four processes: encoding, storage, consolidation and retrieval. We argue that synaptic plasticity is critical for the encoding and intermediate storage of memory traces that are automatically recorded in the hippocampus. These traces decay, but are sometimes retained by a process of cellular consolidation. However, we also argue that HPC synaptic plasticity is not involved in memory retrieval, and is unlikely to be involved in systems-level consolidation that depends on HPC-neocortical interactions, although neocortical synaptic plasticity does play a part. The information that has emerged from the worldwide focus on the mechanisms of induction and expression of plasticity at individual synapses has been very valuable in functional studies. Progress towards a comprehensive understanding of memory processing will also depend on the analysis of these synaptic changes within the context of a wider range of systems-level and cellular mechanisms of neuronal transmission and plasticity.

Functions of cannabinoid receptors in the hippocampus.

Marijuana smoking is recognised to impair human cognition and learning, but the mechanisms by which this occurs are not well characterised. This article focuses exclusively on the hippocampus to review the effects of cannabinoids on hippocampal function and evaluate the evidence that hippocampal cannabinoid receptors play a role in learning and formation of memory. Activation of cannabinoid receptors inhibits release of a variety of neurotransmitters, and modulates a number of intrinsic membrane conductances. Suppression of inhibitory GABAergic synaptic transmission has been repeatedly described, but whether there is also control of excitatory glutamatergic transmission is more controversial. The recognition that the commonly used WIN55,212-2 also acts via non-cannabinoid receptors may help resolve this issue. The involvement of endocannabinoids in depolarisation induced suppression of inhibition (DSI) and the demonstration that activation of metabotropic glutamate receptors can stimulate endocannabinoid release have provided the first insights into the physiological roles of the cannabinoids. Cannabinoids have consistently been reported to inhibit high frequency stimulation induced synaptic long-term potentiation but the experimental design of most behavioural experiments have meant it is not possible to categorically demonstrate a role for hippocampal cannabinoid receptors in learning and memory.

Hematogenous cervical spondylodiscitis after severe burn injury.

A 47-year-old man sustained a 31% TBSA burn injury. In spite of early escharectomy and mesh-graft-transplantation the patient suffered a septicaemic phase in the first week, which was treated by a specific antibiotic. Five weeks after the burn injury a cervical spondylodiscitis was diagnosed. Immediate wound debridement, ventral and dorsal spondylodesis with a tricortical bone-graft from the left iliac crest and titanium plates and specific antibiotic therapy led to the stabilization and healing of the cervical spinal column. The spondylodiscitis was microbiologically proved to be hematogenous after spread of Staphylococcus aureus from the blood in the early septicaemic phase. Swab culture from the burn surface wound, infected vertebrae and blood during the septicaemic phase revealed coagulase positive S. aureus. The aetiology, predisposing factors and management of this rare, but recognized, complication of major burns are discussed. Case features of this patient are compared with the single site's reported case of hematogenous cervical spondylodiscitis after severe burn injury.

Aluminium toxicity in the rat brain: histochemical and immunocytochemical evidence.

Although the neurotoxic actions of aluminium (Al) have been well documented, its contribution to neurodegenerative diseases such as Alzheimer's disease remains controversial. In the present study, we applied histochemical techniques to identify changes induced by intracerebroventricular Al injections (5.4 microg in 5.5 microl, daily over a period of 5 successive days) in the adult rat brain after survival periods of either 1 or 6 weeks. For both Al- and saline-infused controls, no major signs of gross histological changes were evident in cresyl violet-stained sections. Al (as indicated by the fluorescent Morin staining) was concentrated in white matter of the medial striatum, corpus callosum, and cingulate bundle. Immunoreactivity of astrocytes and phagocytic microglia based on glial fibrillary acidic protein and ED1 markers, respectively, revealed a greater inflammatory response in Al-injected animals compared to controls. Damage of the cingulate bundle in Al-treated animals led to a severe anterograde degeneration of cholinergic terminals in cortex and hippocampus, as indicated by acetylcholinesterase labelling. Our data suggest that the enhancement of inflammation and the interference with cholinergic projections may be the modes of action through which Al may cause learning and memory deficits, and contribute to pathological processes in Alzheimer's disease.

Function of the hippocampus in memory formation: desperately seeking resolution.

Despite considerable efforts and successes investigating the function of the hippocampal formation in memory processes, there are still numerous elusive key issues. Some of them will be addressed in this review. We will argue that recent evidence supports hippocampal participation in several memory processes, such as encoding, short-term and long-term consolidation and retrieval. While some processes, for example encoding and short-term consolidation, have been the subject of detailed investigations, at least for specific and repeatedly used behavioural paradigms, there appears to be considerable lack of information with respect to other processes, for example long-term consolidation. Although the existence of long-term consolidation is not at debate here, there is only very fragmented information as to the cellular processes enabling long-term consolidation. Recent ample evidence now suggests a potential role in metabotropic glutamate receptors, and more specifically the phospholipase C-coupled receptor 5, in long-term consolidation. The hyperexpression of receptor protein was limited to CA1 indicating a specific role of this brain region in the consolidation of memories. Future work should further explore this important issue especially since long-term consolidation appears to be a necessity for permanent storage of information, and may thus engage memory mechanism that fail during ageing and dementia.