A schizophrenia risk factor induces marked anatomical deficits at GABAergic-dopaminergic synapses in the rat ventral tegmental area: Essential evidence for new targeted therapies.

To develop new therapies for schizophrenia, evidence accumulated over decades highlights the essential need to investigate the GABAergic synapses that presynaptically influence midbrain dopaminergic neurons. Since current technology restricts these studies to animals, and evidence accumulated in recent decades indicates a developmental origin of schizophrenia, we investigated synaptic changes in male rat offspring exposed to maternal immune activation (MIA), a schizophrenia risk factor. Using a novel combination of lentiviruses, peroxidase-immunogold double labeling, three-dimensional serial section transmission electron microscopy and stereology, we observed clear anatomical alterations in synaptic inputs on dopaminergic neurons in the midbrain posterior ventral tegmental area (pVTA). These changes relate directly to a characteristic feature of schizophrenia: increased dopamine release. In 3-month-old and 14-month-old MIA rats, we found a marked decrease in the volume of presynaptic GABAergic terminals from the rostromedial tegmental nucleus (RMTg) and in the length of the synapses they made, when innervating pVTA dopaminergic neurons. In MIA rats in the long-term, we also discovered a decrease in the volume of the postsynaptic density (PSD) and in the maximum thickness of the PSD at the same synapses. These marked deficits were evident in conventional GABA-dopamine synapses and in synaptic triads that we discovered involving asymmetric synapses that innervated RMTg GABAergic presynaptic terminals, which in turn innervated pVTA dopaminergic neurons. In triads, the PSD thickness of asymmetric synapses was significantly decreased in MIA rats in the long-term cohort. The extensive anatomical deficits provide a potential basis for new therapies targeted at synaptic inputs on midbrain pVTA dopaminergic neurons, in contrast to current striatum-targeted antipsychotic drugs.

Treatment of Neonatal Hypoxic-Ischemic Encephalopathy with Erythropoietin Alone, and Erythropoietin Combined with Hypothermia: History, Current Status, and Future Research.

Perinatal hypoxic-ischemic encephalopathy (HIE) remains a major cause of morbidity and mortality. Moderate hypothermia (33.5 °C) is currently the sole established standard treatment. However, there are a large number of infants for whom this therapy is ineffective. This inspired global research to find neuroprotectants to potentiate the effect of moderate hypothermia. Here we examine erythropoietin (EPO) as a prominent candidate. Neonatal animal studies show that immediate, as well as delayed, treatment with EPO post-injury, can be neuroprotective and/or neurorestorative. The observed improvements of EPO therapy were generally not to the level of control uninjured animals, however. This suggested that combining EPO treatment with an adjunct therapeutic strategy should be researched. Treatment with EPO plus hypothermia led to less cerebral palsy in a non-human primate model of perinatal asphyxia, leading to clinical trials. A recent Phase II clinical trial on neonatal infants with HIE reported better 12-month motor outcomes for treatment with EPO plus hypothermia compared to hypothermia alone. Hence, the effectiveness of combined treatment with moderate hypothermia and EPO for neonatal HIE currently looks promising. The outcomes of two current clinical trials on neurological outcomes at 18-24 months-of-age, and at older ages, are now required. Further research on the optimal dose, onset, and duration of treatment with EPO, and critical consideration of the effect of injury severity and of gender, are also required.

Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology.

Viral vectors were originally developed to deliver genes into host cells for therapeutic potential. However, viral vector use in neuroscience research has increased because they enhance interpretation of the anatomy and physiology of brain circuits compared with conventional tract tracing or electrical stimulation techniques. Viral vectors enable neuronal or glial subpopulations to be labeled or stimulated, which can be spatially restricted to a single target nucleus or pathway. Here we review the use of viral vectors to examine the structure and function of motor and limbic basal ganglia (BG) networks in normal and pathological states. We outline the use of viral vectors, particularly lentivirus and adeno-associated virus, in circuit tracing, optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at gross or ultrastructural levels are reviewed. We explain how optogenetic stimulation and designer drugs used to modulate a distinct pathway and neuronal subpopulation have enhanced our mechanistic understanding of BG function in health and pathophysiology in disease. Finally, we outline how viral vector technology may be applied to neurological and psychiatric conditions to offer new treatments with enhanced outcomes for patients.

Marked differences in the number and type of synapses innervating the somata and primary dendrites of midbrain dopaminergic neurons, striatal cholinergic interneurons, and striatal spiny projection neurons in the rat.

Elucidating the link between cellular activity and goal-directed behavior requires a fuller understanding of the mechanisms underlying burst firing in midbrain dopaminergic neurons and those that suppress activity during aversive or non-rewarding events. We have characterized the afferent synaptic connections onto these neurons in the rat substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA), and compared these findings with cholinergic interneurons and spiny projection neurons in the striatum. We found that the average absolute number of synapses was three to three and one-half times greater onto the somata of dorsal striatal spiny projection neurons than onto the somata of dopaminergic neurons in the SNpc or dorsal striatal cholinergic interneurons. A similar comparison between populations of dopamine neurons revealed a two times greater number of somatic synapses on VTA dopaminergic neurons than SNpc dopaminergic neurons. The percentage of symmetrical, presumably inhibitory, synaptic inputs on somata was significantly higher on spiny projection neurons and cholinergic interneurons compared with SNpc dopaminergic neurons. Synaptic data on the primary dendrites yielded similar significant differences for the percentage of symmetrical synapses for VTA dopaminergic vs. striatal neurons. No differences in the absolute number or type of somatic synapses were evident for dopaminergic neurons in the SNpc of Wistar vs. Sprague-Dawley rat strains. These data from identified neurons are pivotal for interpreting their electrophysiological responses to afferent activity and for generating realistic computer models of neuronal networks of striatal and midbrain dopaminergic function.

Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms.

Lentiviruses have been extensively used as gene delivery vectors since the mid-1990s. Usually derived from the human immunodeficiency virus genome, they mediate efficient gene transfer to non-dividing cells, including neurons and glia in the adult mammalian brain. In addition, integration of the recombinant lentiviral construct into the host genome provides permanent expression, including the progeny of dividing neural precursors. In this review, we describe targeted vectors with modified envelope glycoproteins and expression of transgenes under the regulation of cell-selective and inducible promoters. This technology has broad utility to address fundamental questions in neuroscience and we outline how this has been used in rodents and primates. Combining viral tract tracing with immunohistochemistry and confocal or electron microscopy, lentiviral vectors provide a tool to selectively label and trace specific neuronal populations at gross or ultrastructural levels. Additionally, new generation optogenetic technologies can be readily utilized to analyze neuronal circuit and gene functions in the mature mammalian brain. Examples of these applications, limitations of current systems and prospects for future developments to enhance neuroscience knowledge will be reviewed. Finally, we will discuss how these vectors may be translated from gene therapy trials into the clinical setting.

Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of striatal injury and may lead to cerebral palsy. This study investigated whether delayed administration of bone marrow-derived mesenchymal stem cells (MSCs), at one week after neonatal rat hypoxia-ischemia, was neurorestorative of striatal medium-spiny projection neurons and improved motor function. The effect of a subcutaneous injection of a high-dose, or a low-dose, of MSCs was investigated in stereological studies. Postnatal day (PN) 7 pups were subjected to hypoxia-ischemia. At PN14, pups received treatment with either MSCs or diluent. A subset of high-dose pups, and their diluent control pups, were also injected intraperitoneally with bromodeoxyuridine (BrdU), every 24h, on PN15, PN16 and PN17. This permitted tracking of the migration and survival of neuroblasts originating from the subventricular zone into the adjacent injured striatum. Pups were euthanized on PN21 and the absolute number of striatal medium-spiny projection neurons was measured after immunostaining for DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32), double immunostaining for BrdU and DARPP-32, and after cresyl violet staining alone. The absolute number of striatal immunostained calretinin interneurons was also measured. There was a statistically significant increase in the absolute number of DARPP-32-positive, BrdU/DARPP-32-positive, and cresyl violet-stained striatal medium-spiny projection neurons, and fewer striatal calretinin interneurons, in the high-dose mesenchymal stem cell (MSC) group compared to their diluent counterparts. A high-dose of MSCs restored the absolute number of these neurons to normal uninjured levels, when compared with previous stereological data on the absolute number of cresyl violet-stained striatal medium-spiny projection neurons in the normal uninjured brain. For the low-dose experiment, in which cresyl violet-stained striatal medium-spiny neurons alone were measured, there was a lower statistically significant increase in their absolute number in the MSC group compared to their diluent controls. Investigation of behavior in another cohort of animals showed that delayed administration of a high-dose of bone marrow-derived MSCs, at one week after neonatal rat hypoxia-ischemia, improved motor function on the cylinder test. Thus, delayed therapy with a high- or low-dose of adult MSCs, at one week after injury, is effective in restoring the loss of striatal medium-spiny projection neurons after neonatal rat hypoxia-ischemia and a high-dose of MSCs improved motor function.

Number and type of synapses on the distal dendrite of a rat striatal cholinergic interneuron: a quantitative, ultrastructural study.

Knowledge of the innervation of interneurons within the striatum is critical to determining their role in the functioning of the striatal network. To this end, the synaptic innervation of a distal dendrite of a rat striatal cholinergic interneuron was quantified for the first time. These synaptic data were compared to three other dendrites from rat striatal interneurons and to published data from dendrites in the mammalian cerebral cortex. To label the cholinergic interneurons and their distal dendrites, a male Wistar rat was perfused and the striatum was double-immunolabelled with an antibody to choline acetyltransferase (ChAT) and an antibody to m2 muscarinic receptor. After processing for transmission electron microscopy, a cholinergic interneuron was located and an m2-labelled distal dendrite identified by tracing it through serial ultrathin sections to this double-immunolabelled soma. Two interneuronal distal dendrites in the same tissue, and another from a second rat, were used for comparison. The widths and lengths of the four distal dendrites, the total number and type of synapses, and the number of synapses per mum for each distal dendrite were measured. Symmetric synapses were the most common type on all four dendrites. There were 0.73 synapses per mum on the distal dendrite of the identified striatal cholinergic interneuron. Two other interneuronal dendrites that were positive for the m2 muscarinic receptor antibody showed similar synaptic densities of 0.62 and 0.83 synapses per microm of distal dendrite, respectively. On a third unlabelled interneuronal distal dendrite located in the lateral striatum, there were 2.17 synapses per microm. This interneuron was thought to be a parvalbumin interneuron rather than a calretinin interneuron, which would more likely be medially located. These data suggest that the number of synapses per microm on the distal dendrite of the cholinergic interneuron, and possibly two other cholinergic interneurons, is three times lower than that of a likely parvalbumin interneuron in the rat striatum. The number of synapses per microm of distal dendrite for a striatal cholinergic interneuron is also lower than the published 1.22-3.3 synapses per microm of dendrite for neurons in the mammalian cerebral cortex. Such anatomical data are important for the construction of new generation computer models that are better able to emulate the operation of striatal cholinergic interneurons.

Early motor development is abnormal in complexin 1 knockout mice.

Complexin I expression is dysregulated in a number of neurological diseases including schizophrenia and depression. Adult complexin 1 knockout (Cplx1(-/-)) mice are severely ataxic and show deficits in exploration and emotional reactivity. Here, we evaluated early behavioural development of Cplx1(-/-) mice. Cplx1(-/-) mice showed marked abnormalities. They develop ataxia by post-natal day 7 (P7), and by P21 show marked deficits in tasks requiring postural skills and complex movement. These deficits are consistent with abnormalities in sensory and motor development found in infants that develop schizophrenia in later life. A role for complexin I depletion should be considered in diseases where deficits in early sensory and motor development exist, such as autism and schizophrenia.