Viral labeling of neurons synaptically connected to nucleus accumbens somatostatin interneurons.

The nucleus accumbens, a key brain reward region, receives synaptic inputs from a range of forebrain and brainstem regions. Many of these projections have been established using electrophysiology or fluorescent tract tracing. However, more recently developed viral tracing techniques have allowed for fluorescent labeling of synaptic afferents in a cell type-specific manner. Since the NAc is comprised of multiple cell types, these methods have enabled the delineation of the cell type-specific connectivity of principal medium spiny neurons in the region. The synaptic connectivity of somatostatin interneurons, which account for <5% of the neurons in the region, has been inferred from electrophysiological and immunohistochemical data, but has not yet been visualized using modern viral tracing techniques. Here, we use the pseudorabies virus (PRV)-Introvert-GFP virus, an alphaherpes virus previously shown to label synaptic afferents in a cell type-specific manner, to label first order afferents to NAc somatostatin interneurons. While we find GFP(+) labeling in several well established projections to the NAc, we also observe that several known projections to NAc did not contain GFP(+) cells, suggesting they do not innervate somatostatin interneurons in the region. A subset of the GFP(+) afferents are c-FOS(+) following acute administration of cocaine, showing that NAc somatostatin interneurons are innervated by some cells that respond to rewarding stimuli. These results provide a foundation for future studies aimed toward elucidating the cell type-specific connectivity of the NAc, and identify specific circuits that warrant future functional characterization.

Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP.

Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre.

Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry.

The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.SIGNIFICANCE STATEMENT The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.

Molecular characterization of neuronal cell types based on patterns of projection with Retro-TRAP.

Retro-TRAP (translating ribosome affinity purification) technology enables the synthesis of molecular and neuroanatomical information through the use of transgenic and viral approaches. In contrast to other methods that are used to profile neural circuits such as laser-capture microdissection and FACS, Retro-TRAP is a high-throughput methodology that requires minimal specialized instrumentation. Retro-TRAP uses an anti-GFP ribosomal tag (expressed virally or using transgenesis) to immunoprecipitate translating mRNAs from any population of neurons that express GFP. The protocol detailed here describes the rapid extraction of molecular information from neural circuits in mice using retrograde-tracing GFP-expressing viruses. This approach can be used to identify novel cell types, as well as to molecularly profile cell types for which Cre-driver lines are available, in defined presynaptic loci. The current protocol describes a method for extracting translating mRNA from any neural circuit accessible by stereotaxic injection and manual dissection, and it takes 2-4 weeks. Although it is not described here, this mRNA can then be used in downstream processing applications such as quantitative PCR (qPCR) and high-throughput RNA sequencing to obtain 'molecular connectomic' information.

Molecular profiling of neurons based on connectivity.

The complexity and cellular heterogeneity of neural circuitry presents a major challenge to understanding the role of discrete neural populations in controlling behavior. While neuroanatomical methods enable high-resolution mapping of neural circuitry, these approaches do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of an approach for molecularly profiling projective neurons. We show that ribosomes can be tagged with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from neurons retrogradely labeled with GFP. Using this system, we profiled neurons projecting to the nucleus accumbens. We then used an AAV to selectively profile midbrain dopamine neurons projecting to the nucleus accumbens. By comparing the captured mRNAs from each experiment, we identified a number of markers specific to VTA dopaminergic projection neurons. The current method provides a means for profiling neurons based on their projections.

Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus.

Rapid-eye movement (REM) sleep correlates with neuronal activity in the brainstem, basal forebrain and lateral hypothalamus. Lateral hypothalamus melanin-concentrating hormone (MCH)-expressing neurons are active during sleep, but their effects on REM sleep remain unclear. Using optogenetic tools in newly generated Tg(Pmch-cre) mice, we found that acute activation of MCH neurons (ChETA, SSFO) at the onset of REM sleep extended the duration of REM, but not non-REM, sleep episodes. In contrast, their acute silencing (eNpHR3.0, archaerhodopsin) reduced the frequency and amplitude of hippocampal theta rhythm without affecting REM sleep duration. In vitro activation of MCH neuron terminals induced GABAA-mediated inhibitory postsynaptic currents in wake-promoting histaminergic neurons of the tuberomammillary nucleus (TMN), and in vivo activation of MCH neuron terminals in TMN or medial septum also prolonged REM sleep episodes. Collectively, these results suggest that activation of MCH neurons maintains REM sleep, possibly through inhibition of arousal circuits in the mammalian brain.

Hypothalamic melanin concentrating hormone neurons communicate the nutrient value of sugar.

Sugars that contain glucose, such as sucrose, are generally preferred to artificial sweeteners owing to their post-ingestive rewarding effect, which elevates striatal dopamine (DA) release. While the post-ingestive rewarding effect, which artificial sweeteners do not have, signals the nutrient value of sugar and influences food preference, the neural circuitry that mediates the rewarding effect of glucose is unknown. In this study, we show that optogenetic activation of melanin-concentrating hormone (MCH) neurons during intake of the artificial sweetener sucralose increases striatal dopamine levels and inverts the normal preference for sucrose vs sucralose. Conversely, animals with ablation of MCH neurons no longer prefer sucrose to sucralose and show reduced striatal DA release upon sucrose ingestion. We further show that MCH neurons project to reward areas and are required for the post-ingestive rewarding effect of sucrose in sweet-blind Trpm5(-/-) mice. These studies identify an essential component of the neural pathways linking nutrient sensing and food reward. DOI: http://dx.doi.org/10.7554/eLife.01462.001.

Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons.

Ventral tegmental area (VTA) dopamine neurons in the brain's reward circuit have a crucial role in mediating stress responses, including determining susceptibility versus resilience to social-stress-induced behavioural abnormalities. VTA dopamine neurons show two in vivo patterns of firing: low frequency tonic firing and high frequency phasic firing. Phasic firing of the neurons, which is well known to encode reward signals, is upregulated by repeated social-defeat stress, a highly validated mouse model of depression. Surprisingly, this pathophysiological effect is seen in susceptible mice only, with no apparent change in firing rate in resilient individuals. However, direct evidence--in real time--linking dopamine neuron phasic firing in promoting the susceptible (depression-like) phenotype is lacking. Here we took advantage of the temporal precision and cell-type and projection-pathway specificity of optogenetics to show that enhanced phasic firing of these neurons mediates susceptibility to social-defeat stress in freely behaving mice. We show that optogenetic induction of phasic, but not tonic, firing in VTA dopamine neurons of mice undergoing a subthreshold social-defeat paradigm rapidly induced a susceptible phenotype as measured by social avoidance and decreased sucrose preference. Optogenetic phasic stimulation of these neurons also quickly induced a susceptible phenotype in previously resilient mice that had been subjected to repeated social-defeat stress. Furthermore, we show differences in projection-pathway specificity in promoting stress susceptibility: phasic activation of VTA neurons projecting to the nucleus accumbens (NAc), but not to the medial prefrontal cortex (mPFC), induced susceptibility to social-defeat stress. Conversely, optogenetic inhibition of the VTA-NAc projection induced resilience, whereas inhibition of the VTA-mPFC projection promoted susceptibility. Overall, these studies reveal novel firing-pattern- and neural-circuit-specific mechanisms of depression.

Molecular profiling of activated neurons by phosphorylated ribosome capture.

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

Maintenance of respiratory chain function in mouse hearts with severely impaired mtDNA transcription.

The basal mitochondrial transcription machinery is essential for biogenesis of the respiratory chain and consists of mitochondrial RNA polymerase, mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2. This triad of proteins is sufficient and necessary for mtDNA transcription initiation. Abolished mtDNA transcription caused by tissue-specific knockout of TFAM in the mouse heart leads to early onset of a severe mitochondrial cardiomyopathy with lethality within the first post-natal weeks. Here, we describe a mouse model expressing human TFAM instead of the endogenous mouse TFAM in heart. These rescue mice have severe reduction in mtDNA transcription initiation, but, surprisingly, are healthy at the age of 52 weeks with near-normal steady-state levels of transcripts. In addition, we demonstrate that heavy-strand mtDNA transcription normally terminates at the termination-associated sequence in the control region. This termination is abolished in rescue animals resulting in heavy (H)-strand transcription of the entire control region. In conclusion, we demonstrate here the existence of an unexpected mtDNA transcript stabilization mechanism that almost completely compensates for the severely reduced transcription initiation in rescue hearts. Future elucidation of the underlying molecular mechanism may provide a novel pathway to treat mitochondrial dysfunction in human pathology.

The MitoPark Mouse - an animal model of Parkinson's disease with impaired respiratory chain function in dopamine neurons.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by selective and progressive degeneration of dopamine neurons in the substantia nigra. While most cases are sporadic a few rare familial forms of PD have been described. Several lines of evidence indicate that mitochondrial dysfunction may be involved in the etiology of the disease. Genes found to cause familial Parkinsonism have been linked to mitochondrial function and toxins that inhibit the mitochondrial respiratory chain have been found to cause dopamine cell death. Furthermore, higher numbers of respiratory chain deficient dopamine neurons are found in patients with PD than in age-matched controls. The MitoPark mouse model of PD was designed to directly test the hypothesis that mitochondrial dysfunction in dopamine neurons can cause a progressive parkinsonian phenotype. By cell type-specific inactivation of mitochondrial transcription factor A, a protein essential for mitochondrial DNA expression and maintenance, dopamine neurons were rendered respiratory chain deficient. MitoPark mice recapitulate several features of PD in humans such as adult-onset degeneration of nigrostriatal dopamine circuitry; motor deficits that are ameliorated by L-DOPA administration; progressive course of phenotypic manifestations and neurodegeneration; and altered response to L-DOPA treatment dependent on disease stage. In this review we compare the MitoPark mouse to other genetic or toxin-based rodent models of Parkinson's disease.

The alpha-herpesviruses: molecular pathfinders in nervous system circuits.

Several neuroinvasive viruses can be used to study the mammalian nervous system. In particular, infection by pseudorabies virus (PRV), an alpha-herpesvirus with broad host range, reveals chains of functionally connected neurons in the nervous systems of a variety of mammals. The specificity of PRV trans-neuronal spread has been established in several systems. One attenuated strain, PRV-Bartha, causes a reduced inflammatory response and also spreads only from infected post- to pre-synaptic neurons. We review the basics of PRV tracing and then discuss new developments and novel approaches that have enabled a more detailed understanding of the architecture of the nervous system. As questions and techniques evolve in the field of neuroscience, advances in PRV tracing will certainly follow.

Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons.

Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson's disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD.

Mitochondrial DNA polymerase gamma is essential for mammalian embryogenesis.

Mitochondrial DNA (mtDNA) polymerase gamma (Polg) is a heterodimeric enzyme containing a Pol I-like catalytic core (PolgA) and an accessory subunit. Mutations in POLGA, affecting the stability of mtDNA, have been identified in several human pathologies such as progressive external ophthalmoplegia and Alpers' syndrome. Extensive literature shows mitochondrial toxicity effects nucleoside analogue reverse transcriptase inhibitors used in the treatment of HIV and chronic hepatitis B as a consequence of an inhibitory effect on Polg. We have previously shown that mice with an error-prone version of PolgA accumulate higher levels of somatic mtDNA mutations resulting in a premature aging phenotype. In the present paper, we demonstrate PolgA deficiency in mouse embryos causes an early developmental arrest between embryonic days 7.5 and 8.5 associated with severe mtDNA depletion. Heterozygous knockout mice have half the wild-type levels of PolgA transcripts and a slight reduction in mtDNA levels but develop normally. Surprisingly, amounts of PolgA transcripts in heterozygous knockout mice are increased in response to artificially elevated mtDNA copy number, revealing a possible regulatory link between mtDNA maintenance and PolgA expression. Our results show that Polg indeed is the only DNA polymerase capable of maintaining mtDNA in mammalian mitochondria. In addition, presence of Polg is absolutely essential for the organogenesis during mammalian embryonic development.

Mitochondrial transcription factor A regulates mtDNA copy number in mammals.

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.