Evidence for the differential co-localization of neurokinin-1 receptors with 5-HT receptor subtypes in rat forebrain.

Studies suggest that like selective 5-hydroxytryptamine (5-HT; serotonin) reuptake inhibitors, antagonists at neurokinin-1 receptors (NK(1)Rs) may have antidepressant and anxiolytic properties. NK(1)Rs are present in 5-HT innervated forebrain regions which may provide a common point of interaction between these two transmitter systems. This study aimed to investigate for cellular co-localization between NK(1)Rs and 5-HT receptor subtypes in mood-related brain regions in the rat forebrain. With experiments using fluorescence immunocytochemistry, double-labelling methods demonstrated a high degree of co-localization between NK(1)Rs and 5-HT(1A) receptors in most regions examined. Co-localization was highest in the medial septum (88% NK(1)R expressing cells were 5-HT(1A) receptor-positive) and hippocampal regions (e.g. dentate gyrus, 65%), followed by the lateral/basolateral amygdala (35%) and medial prefrontal cortex (31%). In contrast, co-localization between NK(1)Rs and 5-HT(2A) receptors was infrequent (< 8%) in most areas examined except for the hippocampus (e.g. CA3, 43%). Overall co-localization between NK(1)Rs and 5-HT(1A) receptors was much greater than that between NK(1)Rs and 5-HT(2A) receptors. Thus, these experiments demonstrate a high degree of co-localization between NK(1)Rs and 5-HT(1A) receptors in cortical and limbic regions of the rat forebrain. These findings suggest a novel site of interaction between NK(1)R antagonists and the 5-HT system.

Sustained stress-induced changes in mice as a model for chronic depression.

RATIONALE: Major depression is a chronic disabling disorder, often preceded by stress. Despite emerging clinical interest in mechanisms perpetuating episodes of depression and/or establishing increased vulnerability for relapse, little attention has been paid to address these aspects in experimental models. Here, we studied the long-term neuroadaptive effects of chronic mild stress (CMS) as well as the effectiveness of a course of an antidepressant treatment. METHODS: CMS was applied for 6 weeks, and paroxetine was administered from the third week and continued for 2 weeks thereafter. In order to validate our CMS procedure, we first studied short-term (24 h after CMS) hippocampal cell proliferation and neurogenesis, along with anhedonic-like behaviour. Subsequently, we examined the long-term (one month after CMS) anhedonia, hippocampal neurogenesis, the regulation of c-Fos immunoreactivity and neurotransmitter levels in different areas as well as cortical spine density and hippocampal expression of synaptic proteins. RESULTS: CMS induced a decrease in short-term neurogenesis that was fully recovered in the long term. In addition, CMS-induced lasting anhedonia and region-specific changes in neuronal activity (c-Fos immunoreactivity) and neurotransmitter (glutamate and GABA) levels. Repeated paroxetine reverted these effects with the exception of decreased neuronal activity in the dentate gyrus (DG) and GABA levels in the ventral hippocampus. Moreover, CMS downregulated the GAD65 and VGLUT1 expressions. CONCLUSION: This study shows region-specific long-term neurobiological adaptations induced by CMS and residual hippocampal signs after paroxetine treatment. We propose the use of this model to study molecular mechanisms involved in chronic depression and vulnerability for relapse.

Modeling PD pathogenesis in mice: advantages of a chronic MPTP protocol.

Formidable challenges for Parkinson's disease (PD) research are to understand the processes underlying nigrostriatal degeneration and how to protect dopamine neurons. Fundamental research relies on good animal models that demonstrate the pathological hallmarks and motor deficits of PD. Using a chronic regimen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p) in mice, dopamine cell loss exceeds 60%, extracellular glutamate is elevated, cytoplasmic inclusions are formed and inflammation is chronic. Nevertheless, isradipine, an L-type calcium-channel blocker, attenuates the degeneration. These data support the validity of the MPTP/p model for unravelling the degenerative processes in PD and testing therapies that slow their progress.

Chandelier cartridges in the prefrontal cortex are reduced in isolation reared rats.

Chandelier neurons are a subset of parvalbumin containing cortical interneurons characterised by their preferential targeting of the axon initial segments of pyramidal neurons. They have been the focus of recent interest after evidence that the arrays of boutons are reduced in the prefrontal cortex of schizophrenic patients, post mortem. Since one chandelier neuron may innervate the axon initial segments of several hundred pyramidal neurons, it is hypothesized that their special connectivity might facilitate synchronisation of cortical outputs and play a key role in working memory. Disruption in their function is therefore thought to play a potentially important role in cortically associated symptoms of schizophrenia. Using the isolation rearing animal model of schizophrenia, we examined immunolabelling for GABA-transporter 1, a marker of chandelier cartridges. We show that the numbers of arrays of chandelier axons are reduced by 36% in the ventral prelimbic cortex of isolation-reared rats, compared with their socially-housed litter mates. This mimics findings in the PFC of schizophrenic patients where GAT-1-positive cartridges are reduced by 40% and is the first study to demonstrate changes in chandelier cartridges in an animal model of schizophrenia.

The subiculum comes of age.

The subiculum has long been considered as a simple bidirectional relay region interposed between the hippocampus and the temporal cortex. Recent evidence, however, suggests that this region has specific roles in the cognitive functions and pathological deficits of the hippocampal formation. A group of 20 researchers participated in an ESF-sponsored meeting in Oxford in September, 2005 focusing on the neurobiology of the subiculum. Each brought a distinct expertise and approach to the anatomy, physiology, psychology, and pathologies of the subiculum. Here, we review the recent findings that were presented at the meeting.

NOS-positive local circuit neurons are exclusively axo-dendritic cells both in the neo- and archi-cortex of the rat brain.

Neuronal nitric oxide synthase (nNOS)-containing neurons and axon terminals were examined in the rat somatosensory and temporal neocortex, in the CA3/a-c areas of Ammon's horn and in the hippocampal dentate gyrus. In these areas, only nonpyramidal neurons were labeled with the antibody against nNOS. Previous observations suggested that all nNOS-positive nonpyramidal cells are GABAergic local circuit neurons, which form exclusively symmetric synapses. In agreement with this, nNOS-positive axon terminals in the hippocampal formation formed symmetric synapses exclusively with dendritic shafts. In the neocortex, in contrast, in addition to the nNOS-positive axon terminals that formed synapses with unlabeled spiny and aspiny dendrites and with nNOS-positive aspiny dendrites, a small proportion of the nNOS-positive axon terminals formed symmetric synapses with dendritic spines. These results suggest that nNOS-positive local circuit neurons form a distinct group of axo-dendritic cells displaying slightly different domain specificity in the archi- and neocortex. However, nNOS-positive cells show no target selectivity, because they innervate principal cells and local circuit neurons. Afferents to the NOS-positive cells display neither domain nor target selectivity, because small unlabeled terminals formed synapses with both the soma or dendrites of nNOS-positive neurons and an adjacent unlabeled dendrite or spine in both the hippocampal formation and in neocortex.

A comparison of possible markers for chandelier cartridges in rat medial prefrontal cortex and hippocampus.

Chandelier neurons and their characteristic arrays of axonal terminals, known as cartridges, have been implicated in a variety of psychiatric and neurological disorders including schizophrenia and epilepsy. As a result, these neurons have been extensively examined in the brains of several species using a range of markers. However, these markers have not been systematically compared in a single species for their robustness in labelling chandelier cell cartridges. We have therefore examined several markers, reported to label chandelier arrays in primates, for their capacity to mark these structures in rat medial prefrontal cortex and hippocampus. These studies revealed that cartridge-like structures were labelled by parvalbumin and GAT-1 immunohistochemistry in both medial prefrontal cortex and hippocampus of the rat brain. Additionally, GAD65 immunohistochemistry labelled array-like structures preferentially in the dentate gyrus. In contrast, PSA-NCAM, calbindin and GAD67 immunohistochemistry did not reveal any array-like structures in either region of rat brain. These observations indicate that the various immunological markers previously used to visualise chandelier cell cartridges in primates are not equally efficient in labelling these structures in the rat brain, and that GAT-1 immunohistochemistry is the most robust means of visualising chandelier cell cartridges in the regions examined. These are important considerations for quantitative studies in animal models of neurological disorders where chandelier neurons are implicated.

Astroglial plasticity and glutamate function in a chronic mouse model of Parkinson's disease.

Astrocytes play a major role in maintaining low levels of synaptically released glutamate, and in many neurodegenerative diseases, astrocytes become reactive and lose their ability to regulate glutamate levels, through a malfunction of the glial glutamate transporter-1. However, in Parkinson's disease, there are few data on these glial cells or their regulation of glutamate transport although glutamate cytotoxicity has been blamed for the morphological and functional decline of striatal neurons. In the present study, we use a chronic mouse model of Parkinson's disease to investigate astrocytes and their relationship to glutamate, its extracellular level, synaptic localization, and transport. C57/bl mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). From 4 to 8 weeks after treatment, these mice show a significant loss of dopaminergic terminals in the striatum and a significant increase in the size and number of GFAP-immunopositive astrocytes. However, no change in extracellular glutamate, its synaptic localization, or transport kinetics was detected. Nevertheless, the density of transporters per astrocyte is significantly reduced in the MPTP/p-treated mice when compared to controls. These results support reactive gliosis as a means of striatal compensation for dopamine loss. The reduction in transporter complement on individual cells, however, suggests that astrocytic function may be compromised. Although reactive astrocytes are important for maintaining homeostasis, changes in their ability to regulate glutamate and its associated synaptic functions could be important for the progressive nature of the pathophysiology associated with Parkinson's disease.

A critical review of the development and importance of proteinaceous aggregates in animal models of Parkinson's disease: new insights into Lewy body formation.

The pace of development of new animal models of Parkinson's disease (PD) has increased dramatically in the recent past, primarily because of the identification of the protein, alpha-synuclein, in Lewy bodies in both idiopathic and familial PD. This discovery has allowed the production of transgenic models that incorporate a form of human, mutant alpha-synuclein from rare familial cases, and has enabled the search for Lewy-body-like aggregations of this protein in toxin-induced models. Indeed, alpha-synuclein-positive inclusions, some of which bear strong resemblance to Lewy bodies, have now been recognized and their formation investigated in several different, environmentally-induced and transgenic models. Nevertheless, these data have yet to provide a uniform theory of inclusion pathogenesis for PD. The aim of this review is not only to summarize the findings to date on alpha-synuclein-immunopositive inclusion bodies, including some new information on Lewy bodies, but also provide a concise viewpoint on their origin and formation in animal models. We will provide evidence for a predicted series of intracellular events that underlie inclusion formation. Triggered by oxidative and metabolic stress, chronic, toxin-treated animals, rather than transgenic models transfected with human alpha-synuclein, eventually produce inclusion bodies that most closely resemble early stages of Lewy bodies. Elucidating the common mechanisms in animal models is a first step towards understanding the role of Lewy bodies and their formation in Parkinson's disease.

Quantification of morphological differences in boutons from different afferent populations to the nucleus accumbens.

The nucleus accumbens (Acb) receives convergent glutamatergic inputs from the prefrontal cortex (PFC), central thalamus, basolateral amygdala and the ventral subiculum of the hippocampus. The principal neurons of the nucleus accumbens are modulated by specific sets of convergent afferent inputs, the local circuit neurons also receive a substantial number of glutamatergic inputs, but the full complement of these has yet to be established. The aim of these studies was to define characteristics of the different glutamatergic afferent inputs to the nucleus accumbens that would aid their identification. To enable the characterisation of the glutamatergic inputs to nucleus accumbens neurons we first labelled the four main glutamatergic sources of afferent input to the accumbens with the anterograde tracer biotinylated dextran amine (BDA). Using an unbiased systematic sampling method, the morphological characteristics of their synaptic boutons were measured and assessed at the electron microscopic level. From the criteria assessed, a comparison of the four afferent sources was made, characteristics such as bouton size and vesicle density had significantly different population means, however, the only characteristic that allowed discrimination between the four major glutamatergic afferent to the nucleus accumbens was that of vesicle size. The vesicles in boutons from amygdala were larger than the subiculum which, in turn, were larger than the prefrontal cortex, the thalamus were the smallest in size. The methods used also allow a comparison of the relative frequency of different sized postsynaptic structures targeted, the prefrontal cortex almost exclusively targeted spines whereas the thalamus and the subiculum, in addition to spines, targeted proximal and distal dendrites.

The ultrastructural distribution of alpha-synuclein-like protein in normal mouse brain.

The synaptic protein alpha-synuclein is found throughout the brain, although its function remains ill-defined. Abnormal accumulations of alpha-synuclein have been recognised to be associated with a number of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Nevertheless, little is known about the precise localisation of this protein within the normal brain, information which might contribute to our understanding of its role in both health and disease. We raised an antibody which recognises both human and murine alpha-synuclein and this was used to study the distribution of the protein in the normal mouse brain. We used morphological characteristics to classify the immunopositive presynaptic elements and their targets. We conclude that the protein is present in synaptic boutons of axons with different neurochemical phenotypes but that it is not present in all synaptic terminals. Furthermore, the protein is present in the terminals of neurons such as the dopaminergic neurons of the substantia nigra and the glutamatergic neurons of the hippocampus, cell types which accumulate alpha-synuclein in disease. Nevertheless alpha-synuclein is also found in terminals of neurons which have not been reported to accumulate the protein in neurodegenerative disorders.

Basolateral amygdala efferents to the ventral subiculum preferentially innervate pyramidal cell dendritic spines.

The basolateral amygdala and the ventral subiculum of the hippocampal formation are two of the major limbic-related regions within the brain, both of which project heavily to the nucleus accumbens. The nucleus accumbens is regarded as the limbic-motor interface, in view of these limbic afferent and its somatomotor and autonomic efferent connections. These afferent inputs have been suggested to converge monosynaptically on cells within the accumbens and are hypothesised to play a role in functions such as affective motivational behaviour. Convergence between inputs from the basolateral amygdala and the hippocampus at the level of the accumbens can be demonstrated with electrophysiological recording methods, but these do not conclusively preclude polysynaptic mechanisms. In fact there is a robust reciprocal projection between the basolateral amygdala and the hippocampus, synaptic details of which have not been fully investigated. We examined the synaptic input from the basolateral amygdala to the projection neurons of the subiculum, the spiny pyramidal neurons. We labelled the afferents from basolateral amygdala with a small injection of biotinylated dextran amine, and revealed the anterogradely labeled fibers within the subiculum. The labeled basolateral amygdala fibers were studied with electron microscopy to identify their postsynaptic target structures. With this technique we have demonstrated anatomically that the basolateral amygdala preferentially innervates spiny subiculum neurons, presumed pyramidal projection neurons, although some dendrites and possibly local circuit neurons may be targeted.

Nitric oxide-containing pyramidal neurons of the subiculum innervate the CA1 area.

Neurons and axon terminals containing neuron-specific nitric oxide synthase (nNOS) were examined in the rat subiculum and CA1 area of Ammon's horn. In the subiculum, a large subpopulation of the pyramidal neurons and non-pyramidal cells are immunoreactive for nNOS, whereas in the neighbouring CA1 area of Ammon's horn only non-pyramidal neurons are labelled with the antibody against nNOS. In the pyramidal layer of the subiculum, nNOS-positive axon terminals form both asymmetric and symmetric synapses. In the adjacent CA1 area the nNOS-positive terminals that form symmetric synapses are found in all layers, whereas those terminals that form asymmetric synapses are only in strata radiatum and oriens, but not in stratum lacunosum-moleculare. In both the subiculum and CA1 area, labelled terminals make symmetric synapses only on dendritic shafts, whereas asymmetric synapses are exclusively on dendritic spines. Previous observations demonstrated that all nNOS-positive non-pyramidal cells are GABAergic local circuit neurons, which form exclusively symmetric synapses. We suggest that nNOS-immunoreactive pyramidal cells of the subiculum may innervate neighbouring subicular pyramidal cells and, to a smaller extent, pyramidal cells of the adjacent CA1 area, forming a backward projection between the subicular and hippocampal principal neurons.

Hippocampal and prefrontal cortical inputs monosynaptically converge with individual projection neurons of the nucleus accumbens.

Afferents to the nucleus accumbens from different sources innervate specific areas of the central "core" and peripheral "shell" and are related to each other, at the light microscopical level, in an intricate overlapping and nonoverlapping way. This lack of homogeneity suggests that this region consists of circuits involving emsembles of neurons modulated by specific sets of convergent afferent inputs and abnormal regulation of such ensembles has been implicated in mental disorders. Early extracellular studies suggested that individual Acb neurons might respond to activation of afferents from more than one excitatory input: More recent studies of hippocampal and amygdalar or prefrontal cortical afferents suggest that hippocampal afferents gate the input from the prefrontal cortex or amygdala. Electrophysiological evidence for convergence of excitatory afferents in the Acb is strong and suggests that these pathways are monosynaptic. Nevertheless, this convergence has proved difficult to demonstrate anatomically as a result of the spatial distribution of the afferent inputs on the dendritic tree of the target neurons. To establish whether individual accumbens neurons receive monosynaptic input from pairs of afferents, one projection was labelled anterogradely with Phaseolus vulgaris leucoagglutinin and the second with biotinylated dextran amine (BDA) with Vector slate grey and 3,3'-diaminobenzidine tetrahydrochloride as the chromagens. Accumbens neurons possibly postsynaptic to these afferents, labelled by an in vivo focal injection of BDA, were examined using correlated light and electron microscopy to establish the proximal-distal distribution of labelled afferent synaptic inputs on their dendritic arbours. Individual cells were shown to receive monosynaptic afferent input from both ventral subiculum and prefrontal cortex, providing an anatomical framework for the hippocampal gating of other limbic inputs to the accumbens.