Anatomical and functional characterization of serotonergic neurocircuitry

The serotonin system arises from a small collection of brainstem structures called the raphe nuclei and innervates much of the central nervous system. A relatively small number of serotonin neurons modulate a wide variety of functions, from the basic such as homeostatic regulation of sleep and feeding to more complex roles in mood, motivation and emotion. Furthermore, serotonin signaling is implicated in the etiology and treatment of major depression, and is linked to numerous other mood and neuropsychiatric disorders. However, it has been difficult to assign a defined functional role to serotonin, possibly due to the heterogeneity of the neural population, including molecular, neurochemical, electrophysiological diversity and especially its broad connectivity. The aims of this thesis are to examine the neuroanatomical circuits involving the major serotonergic nuclei, the possible heterogeneity of anatomical wiring including inputs and outputs of the serotonergic population, and connectivity with the basal ganglia structures of the brain. In paper I, we characterize and quantify inputs to serotonergic neurons of the dorsal and median raphe nuclei on a whole-brain scale. We reconstruct whole-brain connectivity patterns by utilizing a genetic strategy for retrograde transsyaptic tracing of direct inputs to genetically-defined serotonergic neurons to uncover previously unidentified or disputed circuits, and provide functional confirmations of direct connections from forebrain regions to serotonergic cells. We characterize functional inputs from the prefrontal cortex, lateral habenula and basal ganglia. In paper II, we determine whether dorsal raphe serotonergic heterogeneity can be characterized by the input-output circuitry of the population. We describe the whole-brain presynaptic inputs to dorsal raphe serotonergic subpopulations that project to either the prefrontal cortex or striatum and contrast these findings with input-output circuitry of other cell types that interact with serotonergic neurons, including midbrain dopaminergic and dorsal raphe GABAegic populations. In paper III, we dissociate the functional connectivity between the different neuronal types of the striatum, a basal ganglia structure that receives serotonergic inputs and also projects to the dorsal raphe. We show that parvalbumin-expressing fast spiking interneurons in the striatum provide direct inhibition onto the projecting medium spiny neurons, which regulate the output to downstream basal ganglia structures, while avoiding other types of neighboring interneurons. In summary, the work of this thesis provides a further step in untangling the heterogeneity of the serotonergic system, by targeting genetically-defined serotonergic neurons of the raphe nuclei and their afferent and efferent connectivity. Ultimately, knowledge of connectivity between genetically defined neuron types will be essential for modeling and understanding circuit function in health and disease

A Whole-Brain Atlas of Inputs to Serotonergic Neurons of the Dorsal and Median Raphe Nuclei

SummaryThe serotonin system is proposed to regulate physiology and behavior and to underlie mood disorders; nevertheless, the circuitry controlling serotonergic neurons remains uncharacterized. We therefore generated a comprehensive whole-brain atlas defining the monosynaptic inputs onto forebrain-projecting serotonergic neurons of dorsal versus median raphe based on a genetically restricted transsynaptic retrograde tracing strategy. We identified discrete inputs onto serotonergic neurons from forebrain and brainstem neurons, with specific inputs from hypothalamus, cortex, basal ganglia, and midbrain, displaying a greater than anticipated complexity and diversity in cell-type-specific connectivity. We identified and functionally confirmed monosynaptic glutamatergic inputs from prefrontal cortex and lateral habenula onto serotonergic neurons as well as a direct GABAergic input from striatal projection neurons. In summary, our findings emphasize the role of hyperdirect inputs to serotonergic neurons. Cell-type-specific classification of connectivity patterns will allow for further functional analysis of the diverse but specific inputs that control serotonergic neurons during behavior

Genetic and environmental determinants for disease risk in subsets of rheumatoid arthritis defined by the anticitrullinated protein/peptide antibody fine specificity profile

OBJECTIVES: To increase understanding of the aetiology and pathogenesis of rheumatoid arthritis (RA), genetic and environmental risk factors for RA subsets, defined by the presence or absence of different anticitrullinated protein/peptide antibodies (ACPAs) targeting citrullinated peptides from α-enolase, vimentin, fibrinogen and collagen type II, were investigated. METHODS: 1985 patients with RA and 2252 matched controls from the EIRA case-control cohort were used in the study. Serum samples were assayed by ELISA for the presence of anticyclic citrullinated peptides (anti-CCP) antibodies and four different ACPA fine specificities. Cross-reactivity between ACPAs was examined by peptide absorption experiments. Genotyping was performed for HLA-DRB1 shared epitope (SE) alleles and the PTPN22 gene, while information regarding smoking was obtained by questionnaire. The association of genetic and environmental risk factors with different subsets of RA was calculated by logistic regression analysis. RESULTS: Limited cross-reactivity was observed between different ACPA fine specificities. In total, 17 RA subsets could be identified based on their different ACPA fine specificity profiles. Large differences in association with genetic and environmental determinants were observed between subsets. The strongest association of HLA-DRB1 SE, PTPN22 and smoking was identified for the RA subset which was defined by the presence of antibodies to citrullinated α-enolase and vimentin. CONCLUSION: This study provides the most comprehensive picture to date of how HLA-DRB1 SE, PTPN22 and smoking are associated with the presence of specific ACPA reactivities rather than anti-CCP levels. The new data will form a basis for molecular studies aimed at understanding disease development in serologically distinct subsets of RA.The Swedish Research CouncilVinnovaKing Gustaf V's 80-year foundationGums&Joints (FP7-Health-2010-261460)MasterSwitch (FP6-Health-2007-2.4.5-12)The Swedish Rheumatic FoundationThe Swedish Council for Working Life and Social ResearchThe IMI program BTCure (115142-2)Publishe

Striatal N-Acetylaspartate Synthetase Shati/Nat8l Regulates Depression-Like Behaviors via mGluR3-Mediated Serotonergic Suppression in Mice

Background: Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3.Methods: Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests.Results: The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes Nacetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor.Conclusions: Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3-mediated functional control of the serotonergic neuronal system

Aversive stimuli drive hypothalamus-to-habenula excitation to promote escape behavior.

A sudden aversive event produces escape behaviors, an innate response essential for survival in virtually all-animal species. Nuclei including the lateral habenula (LHb), the lateral hypothalamus (LH), and the midbrain are not only reciprocally connected, but also respond to negative events contributing to goal-directed behaviors. However, whether aversion encoding requires these neural circuits to ultimately prompt escape behaviors remains unclear. We observe that aversive stimuli, including foot-shocks, excite LHb neurons and promote escape behaviors in mice. The foot-shock-driven excitation within the LHb requires glutamatergic signaling from the LH, but not from the midbrain. This hypothalamic excitatory projection predominates over LHb neurons monosynaptically innervating aversion-encoding midbrain GABA cells. Finally, the selective chemogenetic silencing of the LH-to-LHb pathway impairs aversion-driven escape behaviors. These findings unveil a habenular neurocircuitry devoted to encode external threats and the consequent escape; a process that, if disrupted, may compromise the animal's survival

Mice lacking NMDA receptors in parvalbumin neurons display normal depression-related behavior and response to antidepressant action of NMDAR antagonists.

The underlying circuit imbalance in major depression remains unknown and current therapies remain inadequate for a large group of patients. Discovery of the rapid antidepressant effects of ketamine--an NMDA receptor (NMDAR) antagonist--has linked the glutamatergic system to depression. Interestingly, dysfunction in the inhibitory GABAergic system has also been proposed to underlie depression and deficits linked to GABAergic neurons have been found with human imaging and in post-mortem material from depressed patients. Parvalbumin-expressing (PV) GABAergic interneurons regulate local circuit function through perisomatic inhibition and their activity is NMDAR-dependent, providing a possible link between NMDAR and the inhibitory system in the antidepressant effect of ketamine. We have therefore investigated the role of the NMDAR-dependent activity of PV interneurons for the development of depression-like behavior as well as for the response to rapid antidepressant effects of NMDAR antagonists. We used mutant mice lacking NMDA neurotransmission specifically in PV neurons (PV-Cre+/NR1f/f) and analyzed depression-like behavior and anhedonia. To study the acute and sustained effects of a single NMDAR antagonist administration, we established a behavioral paradigm of repeated exposure to forced swimming test (FST). We did not observe altered behavioral responses in the repeated FST or in a sucrose preference test in mutant mice. In addition, the behavioral response to administration of NMDAR antagonists was not significantly altered in mutant PV-Cre+/NR1f/f mice. Our results show that NMDA-dependent neurotransmission in PV neurons is not necessary to regulate depression-like behaviors, and in addition that NMDARs on PV neurons are not a direct target for the NMDAR-induced antidepressant effects of ketamine and MK801

Sucrose preference test in animals lacking NMDAR in PV neurons.

<p>(A) Outline of the water intake and the sucrose preference test in PV-Cre+/NR1f/f and NR1f/f mice. (B) Water intake test (WIT) in mice lacking NMDAR specifically in PV neurons (PV-Cre+/NR1f/f, <i>red bars</i>, n = 9) compared with control mice (NR1f/f, <i>black bars</i>, n = 6). WIT was run for 24 h. Results (gr) are presented as mean values ±SEM (p>0,05;Student's t-test). (C) Sucrose preference test (SPT) in mice lacking NMDAR specifically in PV neurons (PV-Cre+/NR1f/f, <i>red bars</i>, n = 9) compared with control mice (NR1f/f, <i>black bars</i>, n = 6). SPT was run for 72 h. Results (sucrose intake/total intake ×100) are presented as mean values ±SEM (p>0,05;Student's t-test). Both genotypes display similar behavioral response.</p

Loss of NMDAR in PV neurons does not affect behavior in the repeated FST paradigm.

<p>Immobility of mice lacking NMDAR specifically in PV interneurons (PV-Cre+/NR1f/f,<i>red bars</i>) and control mice (NR1f/f, <i>black bars</i>) during repeated FST. (A) Outline of the repeated FST protocol performed over several days. PV-Cre+/NR1f/f and NR1f/f mice were injected at time 0 with saline (arrow) and every mouse was subjected to FST 30 min, 24 h, 48 h, 72 h and 96 h later. After 4 days of rest in their home cages mice were retested in the FST once again (i.e. 240 h after saline injection). (B) Immobility time is stable after repeated FST in both genotypes. Results (sec/4 min) are presented as mean values ±SEM. Immobility time for NR1f/f mice (<i>black bars</i>, n = 10) is:185±5 (30 min), 205±6 (24 h), 209±8 (48 h), 214±5 (72 h), 187±13 (96 h) and 174±16 (240 h). Immobility time for PV-Cre+/NR1f/f mice (<i>red bars</i>, n = 10) is: 186±5 (30 min), 203±7 (24 h), 207±6 (48 h), 208±4 (72 h), 191±10 (96 h) and 175±7 (240 h).</p

Mice display stable behavior in a repeated FST paradigm.

<p>(A) Outline of the repeated FST protocol performed over several days. C57BL/6N mice (n = 12) were injected at time 0 with saline (arrow) and every mouse was subjected to FST 30 min, 24 h, 48 h, 72 h and 96 h later. After 4 days of rest in their home cages mice were retested in the FST once again (i.e. 240 h after saline injection). (B) Results (sec/4 min) are presented as mean values ±SEM. Immobility time is stable after repeated FST: 161±15 (30 min); 173±17 (24 h); 199±12 (48 h); 208±9 (72 h); 186±9 (96 h) and 183±9 (240 h) (p>0.05, one way ANOVA).</p

MK801-induced antidepressant-like behavioral effects in the FST.

<p>MK801-induced immobility and swimming time in C57BL/6N mice (A,B) and in mice lacking NMDAR specifically in PV neurons (PV-Cre+/NR1f/f, <i>red bars</i>) compared with control (NR1f/f, <i>black bars</i>) (C,D). (A) Immobility of C57BL/6N mice in repeated FST 30 min, 24 h and 1 week after acute treatment with 0.1 mg/kg MK801 (<i>white bars</i>, n = 10) or water (<i>black bars</i>, n = 10). MK801 induces rapid antidepressant-like responses to a lesser extent than ketamine 30 min after treatment. The antidepressant effect of MK801 is abolished 24 h and 1 week after treatment. MK801-induced immobility time is: 220±3 water and 171±17 MK801 (30 min) (p<0.05 Student's t-test); 224±5 water and 223±3 MK801 (24 h) (p>0.05 Student's t-test); 197±10 water and 215±7 MK801 (1 week) (p>0.05 Student's t-test). (B) Swimming scored in the experiment in (A). MK801-induced swimming time is: 20±3 water and 68±17 MK801 (30 min) (p<0.05 Student's t-test); 15±4 water and 16±3 MK801 (24 h) (p>0.05 Student's t-test); 42±10 water and 24±7 MK801 (1week) (p>0.05 Student's t-test). (C) Immobility of PV-Cre+/NR1f/f (<i>red bars</i>) and NR1f/f (<i>black bars</i>) mice in repeated FST 30 min and 24 h after acute treatment with 0.1 mg/kg MK801. Immobility time 30 min and 24 h after MK801 is: 65±16 and 104±13 (NR1f/f <i>black bars</i>, n = 13); 54±16 and 103±16 (PV-Cre+/NR1f/f <i>red bars</i>, n = 14), respectively. (D) Swimming scored in the experiment in (C). Swimming time 30 min and 24 h after MK801 is: 168±15 and 135±16 (NR1f/f <i>black bars</i>, n = 13); 183±15 and 130±14 (PV-Cre+/NR1f/f <i>red bars</i>, n = 14), respectively. All time points p>0,05 (Student's t-test). Both genotypes display similar behavioral response to the MK801 treatment. Results (sec/4 min) are presented as mean values ±SEM.</p