Susan Totterdell

Mouse model of Parkinsonism: a comparison between subacute MPTP and chronic MPTP/probenecid treatment

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used to induce an animal model of Parkinsonism. The conventional mouse model, which usually involves acute or subacute injections of MPTP, results in a significant but reversible loss of dopaminergic functions. We have developed an alternative mouse model, in which co-administration of MPTP with probenecid results in the chronic loss of striatal dopamine for at least 6 months after cessation of treatment. In the present study, we compare the neurochemical, morphological and behavioral changes that occur in this alternative, chronic model with those in the conventional, subacute model. In the chronic model, we demonstrate an almost 80% loss of striatal dopamine and dopamine uptake 6 months after withdrawal from treatment. The neurochemical signs match unbiased stereological measures that demonstrate gradual loss of substantia nigra neurons. Rotarod performance further substantiates these findings by showing a progressive decline in motor performance. Based on the comparisons made in this study in mice, the chronic MPTP/probenecid model shows considerable improvements over the conventional, subacute MPTP model. The sustained alterations in the nigrostriatal pathway resemble the cardinal signs of human Parkinsons disease and suggest that this chronic mouse model is potentially useful to study the pathophysiology and mechanisms of Parkinsonism. It should also prove useful for the development of neuroprotection strategies.

Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson’s disease

We have detected granular and filamentous inclusions that are alpha-synuclein- and ubiquitin-immunoreactive in the cytoplasm of dopaminergic and cortical neurons of C57/black mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid. The immunoreactive aggregates only become evident several weeks after large-scale dopaminergic cell death and a downregulation of alpha-synuclein gene expression. Numerous lipofuscin granules accumulate alpha-synuclein in the nigral and limbic cortical neurons of treated mice. These data provide evidence that insoluble proteins, such as alpha-synuclein, build up as granular and filamentous inclusions in dopaminergic neurons that survive the initial toxic MPTP insult. They further suggest that defective protein degradation rather than altered gene expression underlies deposition of alpha-synuclein and that abundant lysosomal compartments are present to seal off the potentially toxic material.

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. J. Comp. Neurol. 446:151–165, 2002.

Individual nucleus accumbens-projection neurons receive both basolateral amygdala and ventral subicular afferents in rats

The nucleus accumbens is regarded as the limbic-motor interface, in view of its limbic afferent and somatomotor and autonomic efferent connections. Within the accumbens, there appear to be specific areas in which limbic afferent fibres, derived from the hippocampus and the amygdala, overlap. These afferent inputs have been suggested to converge monosynaptically on cells within the accumbens and are hypothesized to play a role in paradigms such as conditioned place preference. Convergence between inputs from basolateral amygdala and hippocampus can be demonstrated with electrophysiological recording methods, but these do not conclusively preclude polysynaptic mechanisms. We examined the synaptic input to the projection neurons of the accumbens, the medium-sized densely spiny neurons. We labelled the projection neurons with a small injection of biotinylated dextran amine into the accumbens, and the afferents from the basolateral amygdala and ventral subiculum of the hippocampus with injections of biotinylated dextran amine and Phaseolus vulgaris-leucoagglutinin respectively, and revealed the anterogradely labelled fibres with different chromogens. The labelled accumbens-projection neurons were studied with correlated light and electron microscopy for identified monosynaptic inputs. With this technique we have demonstrated anatomically that monosynaptic convergence between the ventral subicular region of the hippocampus and the basolateral region of the amygdala occurs at the level of the proximal as well as distal dendrites. Finally, we suggest that these anatomical arrangements may represent the framework for the integrative role that has been assigned to the accumbens.

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

Formidable challenges for Parkinsons 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.

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1)

Three isoforms of a vesicular glutamate transporter (VGLUT1–3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down‐regulation of VGLUT1 transporter on anxiety, depressive‐like behaviour and learning. The behavioural phenotype of VGLUT1‐heterozygous mice (C57BL/6) was compared to wild‐type (WT) littermates. Moreover, VGLUT1–3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1‐heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light–dark exploration test and depressive‐like behaviour in the forced swimming test: no differences were shown in the elevated plus‐maze model of anxiety. In the novel object recognition test, VGLUT1+/– mice showed normal short‐term but impaired long‐term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35–45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1‐mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.

Morphology and distribution of electrophysiologically defined classes of pyramidal and nonpyramidal neurons in rat ventral subiculum in vitro.

Intracellular electrophysiological recordings were made from 210 ventral subicular neurons in rat brain slices. Recordings here classified as burst‐firing or nonburst‐firing. Eighteen burst‐firing neurons were filled with Neurobiotin, and all had pyramidal morphology. Nine of these recordings were made from intrinsically burst‐firing (IB) cell bodies, and nine were made from burst‐firing dendrites (BD). Twelve nonburst‐firing neurons were also filled with Neurobiotin. Eight were regular spiking (RS) and had pyramidal morphology, four were fast spiking (FS) and nonpyramidal. Additional electrophysiological parameters distinguished IB from BD, RS from FS, and RS from IB recordings.

Ultrastructural features of the nitric oxide synthase-containing interneurons in the nucleus accumbens and their relationship with tyrosine hydroxylase-containing terminals.

The ultrastructural features of neuronal nitric oxide synthase (NOS) ‐immunoreactive interneurons of rat nucleus accumbens shell and core were studied and compared. The NOS‐containing subpopulation displayed characteristics similar to those previously described for nicotinamide adenine dinucleotide phosphate diaphorase‐, neuropeptide Y, or somatostatin‐containing striatal neurons, but also showed properties not previously associated with them, particularly the formation of both asymmetric and symmetric synaptic junctions. Inputs derived mainly from unlabeled terminals, but some contacts were made by NOS‐immunolabeled terminals, by means of asymmetric synapses. Immunopositive endings that formed symmetric synapses were mainly onto dendritic shafts, whereas those that formed asymmetric synapses targeted spine heads. Morphometric analysis revealed that the core and shell NOS‐stained neurons had subtly different innervation patterns and that immunostained terminals were significantly larger in the shell. A parallel investigation explored synaptic associations with dopaminergic innervation identified by labeling with an antibody against tyrosine hydroxylase (TH). In both shell and core, TH‐positive boutons formed symmetric synapses onto NOS‐containing dendrites, and in the core, TH‐ and NOS‐immunolabeled terminals converged on both a single spiny dendrite and a spine. These results suggest that, in the rat nucleus accumbens, NOS‐containing neurons may be further partitioned into subtypes, with differing connectivities in shell and core regions. These NOS‐containing neurons may be influenced by a dopaminergic input. Recent studies suggest that nitric oxide potentiates dopamine release and the current study identifies the medium‐sized, densely spiny neurons as a possible site of such an interaction. J. Comp. Neurol. 431:139–154, 2001.

Microcircuits in nucleus accumbens’ shell and core involved in cognition and reward

Nucleus accumbens subserves numerous adaptive and goal-directed behaviors. The anatomical substrates for such are the medium spiny projection neurons, at least four different subtypes of local circuit neurons, and inputs from limbic cortical centers and brainstem monoamines. In this review, we show how microcircuits in two parts of the nucleus—the core and caudomedial shell or septal pole— differ subtly in their connections yet permit quite different behaviors. We further show how small differences in synaptic wiring, especially in relation to the local circuit neurons, can facilitate or suppress behavioral activity. Finally, we consider some important organizational principles that underlie cognitive, emotional, and rewarding behaviors.

Differences in the laminar origin of projections from the medial prefrontal cortex to the nucleus accumbens shell and core regions in the rat

The medial prefrontal cortex (mPFC) projects to the nucleus accumbens shell, core and rostral pole. In this retrograde tract-tracing study of rat mPFC to nucleus accumbens projection neurons, the advantages of Neurobiotin are utilised in order to reveal the detailed morphology of labelled projection cells, and to permit an examination of the laminar projections to shell and core compartments The retrogradely transported Neurobiotin was found in somata, proximal and distal dendrites of neurons that project from the mPFC to the nucleus accumbens. The morphology of these projection neurons was revealed in great detail and confirmed that the projection arises wholly from pyramidal cells. Interestingly, it was also found that retrogradely labelled neurons were exclusively located in prelimbic and infralimbic regions in layers V and VI, after shell injections, but also in layer II following core sites. This observation may reflect possibly different roles for cortical laminae on the nucleus accumbens.