Disease-specific selective vulnerability and neuroimmune pathways in dementia revealed by single cell genomics.

The development of successful therapeutics for dementias requires an understanding of their shared and distinct molecular features in the human brain. We performed single-nuclear RNAseq and ATACseq in Alzheimer disease (AD), Frontotemporal degeneration (FTD), and Progressive Supranuclear Palsy (PSP), analyzing 40 participants, yielding over 1.4M cells from three brain regions ranging in vulnerability and pathological burden. We identify 35 shared disease-associated cell types and 14 that are disease-specific, replicating those previously identified in AD. Disease - specific cell states represent molecular features of disease-specific glial-immune mechanisms and neuronal vulnerability in each disorder, layer 4/5 intra-telencephalic neurons in AD, layer 2/3 intra-telencephalic neurons in FTD, and layer 5/6 near-projection neurons in PSP. We infer intrinsic disease-associated gene regulatory networks, which we empirically validate by chromatin footprinting. We find that causal genetic risk acts in specific neuronal and glial cells that differ across disorders, primarily non-neuronal cells in AD and specific neuronal subtypes in FTD and PSP. These data illustrate the heterogeneous spectrum of glial and neuronal composition and gene expression alterations in different dementias and identify new therapeutic targets by revealing shared and disease-specific cell states.

Tuberous sclerosis complex is associated with a novel human tauopathy.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder leading to epilepsy, developmental delay, and neurobehavioral dysfunction. The syndrome is caused by pathogenic variants in TSC1 (coding for hamartin) or TSC2 (coding for tuberin). Recently, we reported a progressive frontotemporal dementia-like clinical syndrome in a patient with a mutation in TSC1, but the neuropathological changes seen in adults with TSC with or without dementia have yet to be systematically explored. Here, we examined neuropathological findings in adults with TSC (n = 11) aged 30-58 years and compared them to age-matched patients with epilepsy unrelated to TSC (n = 9) and non-neurological controls (n = 10). In 3 of 11 subjects with TSC, we observed a neurofibrillary tangle-predominant "TSC tauopathy" not seen in epilepsy or non-neurological controls. This tauopathy was observed in the absence of pathological amyloid beta, TDP-43, or alpha-synuclein deposition. The neurofibrillary tangles in TSC tauopathy showed a unique pattern of post-translational modifications, with apparent differences between TSC1 and TSC2 mutation carriers. Tau acetylation (K274, K343) was prominent in both TSC1 and TSC2, whereas tau phosphorylation at a common phospho-epitope (S202) was observed only in TSC2. TSC tauopathy was observed in selected neocortical, limbic, subcortical, and brainstem sites and showed a 3-repeat greater than 4-repeat tau isoform pattern in both TSC1 and TSC2 mutation carriers, but no tangles were immunolabeled with MC1 or p62 antibodies. The findings suggest that individuals with TSC are at risk for a unique tauopathy in mid-life and that tauopathy pathogenesis may involve TSC1, TSC2, and related molecular pathways.

Salience Network Atrophy Links Neuron Type-Specific Pathobiology to Loss of Empathy in Frontotemporal Dementia.

Each neurodegenerative syndrome reflects a stereotyped pattern of cellular, regional, and large-scale brain network degeneration. In behavioral variant of frontotemporal dementia (bvFTD), a disorder of social-emotional function, von Economo neurons (VENs), and fork cells are among the initial neuronal targets. These large layer 5 projection neurons are concentrated in the anterior cingulate and frontoinsular (FI) cortices, regions that anchor the salience network, a large-scale system linked to social-emotional function. Here, we studied patients with bvFTD, amyotrophic lateral sclerosis (ALS), or both, given that these syndromes share common pathobiological and genetic factors. Our goal was to determine how neuron type-specific TAR DNA-binding protein of 43 kDa (TDP-43) pathobiology relates to atrophy in specific brain structures and to loss of emotional empathy, a cardinal feature of bvFTD. We combined questionnaire-based empathy assessments, in vivo structural MR imaging, and quantitative histopathological data from 16 patients across the bvFTD/ALS spectrum. We show that TDP-43 pathobiology within right FI VENs and fork cells is associated with salience network atrophy spanning insular, medial frontal, and thalamic regions. Gray matter degeneration within these structures mediated loss of emotional empathy, suggesting a chain of influence linking the cellular, regional/network, and behavioral levels in producing signature bvFTD clinical features.

Evidence of corticofugal tau spreading in patients with frontotemporal dementia.

Common neurodegenerative diseases feature progressive accumulation of disease-specific protein aggregates in selectively vulnerable brain regions. Increasing experimental evidence suggests that misfolded disease proteins exhibit prion-like properties, including the ability to seed corruptive templating and self-propagation along axons. Direct evidence for transneuronal spread in patients, however, remains limited. To test predictions made by the transneuronal spread hypothesis in human tissues, we asked whether tau deposition within axons of the corticospinal and corticopontine pathways can be predicted based on clinical syndromes and cortical atrophy patterns seen in frontotemporal lobar degeneration (FTLD). Sixteen patients with Pick's disease, 21 with corticobasal degeneration, and 3 with FTLD-MAPT were included, spanning a range of clinical syndromes across the frontotemporal dementia (FTD) spectrum. Cortical involvement was measured using a neurodegeneration score, a tau score, and a composite score based on semiquantitative ratings and complemented by an MRI-based cortical atrophy W-map based on antemortem imaging. Midbrain cerebral peduncle and pontine base descending fibers were divided into three subregions, representing prefrontopontine, corticospinal, and parieto-temporo-occipital fiber pathways. Tau area fraction was calculated in each subregion and related to clinical syndrome and cortical measures. Within each clinical syndrome, there were predicted relationships between cortical atrophy patterns and axonal tau deposition in midbrain cerebral peduncle and pontine base. Between syndromes, contrasting and predictable patterns of brainstem axonal tau deposition emerged, with, for example, greater tau in prefrontopontine fibers in behavioral variant FTD and in corticospinal fibers in corticobasal syndrome. Finally, semiquantitative and quantitative cortical degeneration scores predicted brainstem axonal tau deposition based on anatomical principles. Taken together, these findings provide important human evidence in support of axonal tau spreading in patients with specific forms of tau-related neurodegeneration.

Preferential tau aggregation in von Economo neurons and fork cells in frontotemporal lobar degeneration with specific MAPT variants.

Tau aggregation is a hallmark feature in a subset of patients with frontotemporal dementia (FTD). Early and selective loss of von Economo neurons (VENs) and fork cells within the frontoinsular (FI) and anterior cingulate cortices (ACC) is observed in patients with sporadic behavioral variant FTD (bvFTD) due to frontotemporal lobar degeneration (FTLD), including FTLD with tau inclusions (FTLD-tau). Recently, we further showed that these specialized neurons show preferential aggregation of TDP-43 in FTLD-TDP. Whether VENs and fork cells are prone to tau accumulation in FTLD-tau remains unclear, and no previous studies of these neurons have focused on patients with pathogenic variants in the gene encoding microtubule-associated protein tau (FTLD-tau/MAPT). Here, we examined regional profiles of tau aggregation and neurodegeneration in 40 brain regions in 8 patients with FTLD-tau/MAPT and 7 with Pick's disease (PiD), a sporadic form of FTLD-tau that often presents with bvFTD. We further qualitatively assessed the cellular patterns of frontoinsular tau aggregation in FTLD-tau/MAPT using antibodies specific for tau hyperphosphorylation, acetylation, or conformational change. ACC and mid-insula were among the regions most affected by neurodegeneration and tau aggregation in FTLD-tau/MAPT and PiD. In these two forms of FTLD-tau, severity of regional neurodegeneration and tau protein aggregation were highly correlated across regions. In FTLD-tau/MAPT, VENs and fork cells showed disproportionate tau protein aggregation in patients with V337 M, A152T, and IVS10 + 16 variants, but not in patients with the P301L variant. As seen in FTLD-TDP, our data suggest that VENs and fork cells represent preferentially vulnerable neuron types in most, but not all of the MAPT variants we studied.

Neuropathological correlates of structural and functional imaging biomarkers in 4-repeat tauopathies.

Neurodegenerative dementia syndromes are characterized by spreading of pathological protein deposition along syndrome-specific neural networks. Structural and functional MRI measures can assess the integrity of these networks and have been proposed as biomarkers of disease progression for clinical trials. The relationship between in vivo imaging measures and pathological features, at the single subject level, remains largely unknown. Patient-specific maps of atrophy and seed-based intrinsic connectivity disruption, as compared to normal controls, were obtained for 27 patients subsequently diagnosed with progressive supranuclear palsy (n = 16, seven males, age at death 68.9 ± 6.0 years, imaging-to-pathology interval = 670.2 ± 425.1 days) or corticobasal degeneration (n = 11, two males, age at death 66.7 ± 5.4 years, imaging-to-pathology interval = 696.2 ± 482.2 days). A linear mixed effect model with crossed random effects was used to test regional and single-subject level associations between post-mortem regional measures of neurodegeneration and tau inclusion burden, on the one hand, and regional volume loss and seed-based intrinsic connectivity reduction, on the other. A significant association was found between tau inclusion burden and in vivo volume loss, at the regional level and independent of neurodegeneration severity, in both progressive supranuclear palsy [n = 340 regions; beta 0.036; 95% confidence interval (CI): 0.001, 0.072; P = 0.046] and corticobasal degeneration (n = 215 regions; beta 0.044; 95% CI: 0.009, 0.079; P = 0.013). We also found a significant association between post-mortem neurodegeneration and in vivo volume loss in both progressive supranuclear palsy (n = 340 regions; beta 0.155; 95% CI: 0.061, 0.248; P = 0.001) and corticobasal degeneration (n = 215 regions; beta 0.277; 95% CI: 0.104, 0.450; P = 0.002). We found a significant association between regional neurodegeneration and intrinsic connectivity dysfunction in corticobasal degeneration (n = 215 regions; beta 0.074; 95% CI: 0.005, 0.143; P = 0.035), but no other associations between post-mortem measures of tauopathy and intrinsic connectivity dysfunction reached statistical significance. Our data suggest that in vivo structural imaging measures reflect independent contributions from neurodegeneration and tau burden in progressive supranuclear palsy and corticobasal degeneration. Seed-based measures of intrinsic connectivity dysfunction showed less reliable predictive value when used as in vivo biomarkers of tauopathy. The findings provide important guidance for the use of imaging biomarkers as indirect in vivo assays of microscopic pathology.

Neurons selectively targeted in frontotemporal dementia reveal early stage TDP-43 pathobiology.

TAR DNA-binding protein 43 (TDP-43) aggregation is the most common pathological hallmark in frontotemporal dementia (FTD) and characterizes nearly all patients with motor neuron disease (MND). The earliest stages of TDP-43 pathobiology are not well-characterized, and whether neurodegeneration results from TDP-43 loss-of-function or aggregation remains unclear. In the behavioral variant of FTD (bvFTD), patients undergo selective dropout of von Economo neurons (VENs) and fork cells within the frontoinsular (FI) and anterior cingulate cortices. Here, we examined TDP-43 pathobiology within these vulnerable neurons in the FI across a clinical spectrum including 17 patients with sporadic bvFTD, MND, or both. In an exploratory analysis based on our initial observations, we further assessed ten patients with C9orf72-associated bvFTD/MND. VENs and fork cells showed early, disproportionate TDP-43 aggregation that correlated with anatomical and clinical severity, including loss of emotional empathy. The presence of a TDP-43 inclusion was associated with striking nuclear and somatodendritic atrophy. An intriguing minority of neurons lacked detectable nuclear TDP-43 despite the apparent absence of a cytoplasmic TDP-43 inclusion. These cells showed neuronal atrophy comparable to inclusion-bearing neurons, suggesting that the loss of nuclear TDP-43 function promotes neurodegeneration, even when TDP-43 aggregation is inconspicuous or absent.

Early vs late age at onset frontotemporal dementia and frontotemporal lobar degeneration.

OBJECTIVE: To examine clinicopathologic correlations in early vs late age at onset frontotemporal dementia (FTD) and frontotemporal lobar degeneration (FTLD). METHODS: All patients were clinically evaluated and prospectively diagnosed at the UCSF Memory and Aging Center. Two consecutive series were included: (1) patients with a clinically diagnosed FTD syndrome who underwent autopsy (cohort 1) and (2) patients with a primary pathologic diagnosis of FTLD, regardless of the clinical syndrome (cohort 2). These series were divided by age at symptom onset (cutoff 65 years). RESULTS: In cohort 1, 48 (25.3%) were 65 years or older at symptom onset. Pathologic causes of behavioral variant FTD (bvFTD) were similar in the early age at onset (EO) and late age at onset (LO) bvFTD groups. In corticobasal syndrome (CBS), however, the most common pathologic substrate differed according to age at onset: progressive supranuclear palsy (42.9%) in LO-CBS and Alzheimer disease (AD; 40.7%) in EO-CBS. In cohort 2, 57 (28.4%) were classified as LO-FTLD. Regarding FTLD major molecular classes, FTLD with transactive response DNA-binding protein of 43 kDa was most common in EO-FTLD (44.4%), whereas FTLD-tau (58.3%) was most common in LO-FTLD. Antemortem diagnosis of a non-FTD syndrome, usually AD-type dementia, was more frequent in LO-FTLD than EO-FTLD (19.3% vs 7.7%, p = 0.017). LO-FTLD was also associated with more prevalent comorbid pathologic changes. Of these, moderate to severe AD neuropathologic change and argyrophilic grain disease were overrepresented among patients who received an antemortem diagnosis of AD-type dementia. CONCLUSION: Patients with FTD and FTLD often develop symptoms after age 65, and age at onset represents an important consideration when making antemortem neuropathologic predictions.

Von Economo Neurons and Fork Cells: A Neurochemical Signature Linked to Monoaminergic Function.

The human anterior cingulate and frontoinsular cortices are distinguished by 2 unique Layer 5 neuronal morphotypes, the von Economo neurons (VENs) and fork cells, whose biological identity remains mysterious. Insights could impact research on diverse neuropsychiatric diseases to which these cells have been linked. Here, we leveraged the Allen Brain Atlas to evaluate mRNA expression of 176 neurotransmitter-related genes and identified vesicular monoamine transporter 2 (VMAT2), gamma-aminobutyric acid (GABA) receptor subunit θ (GABRQ), and adrenoreceptor α-1A (ADRA1A) expression in human VENs, fork cells, and a minority of neighboring Layer 5 neurons. We confirmed these results using immunohistochemistry or in situ hybridization. VMAT2 and GABRQ expression was absent in mouse cerebral cortex. Although VMAT2 is known to package monoamines into synaptic vesicles, in VENs and fork cells its expression occurs in the absence of monoamine-synthesizing enzymes or reuptake transporters. Thus, VENs and fork cells may possess a novel, uncharacterized mode of cortical monoaminergic function that distinguishes them from most other mammalian Layer 5 neurons.

Clinicopathological correlations in behavioural variant frontotemporal dementia.

Accurately predicting the underlying neuropathological diagnosis in patients with behavioural variant frontotemporal dementia (bvFTD) poses a daunting challenge for clinicians but will be critical for the success of disease-modifying therapies. We sought to improve pathological prediction by exploring clinicopathological correlations in a large bvFTD cohort. Among 438 patients in whom bvFTD was either the top or an alternative possible clinical diagnosis, 117 had available autopsy data, including 98 with a primary pathological diagnosis of frontotemporal lobar degeneration (FTLD), 15 with Alzheimer's disease, and four with amyotrophic lateral sclerosis who lacked neurodegenerative disease-related pathology outside of the motor system. Patients with FTLD were distributed between FTLD-tau (34 patients: 10 corticobasal degeneration, nine progressive supranuclear palsy, eight Pick's disease, three frontotemporal dementia with parkinsonism associated with chromosome 17, three unclassifiable tauopathy, and one argyrophilic grain disease); FTLD-TDP (55 patients: nine type A including one with motor neuron disease, 27 type B including 21 with motor neuron disease, eight type C with right temporal lobe presentations, and 11 unclassifiable including eight with motor neuron disease), FTLD-FUS (eight patients), and one patient with FTLD-ubiquitin proteasome system positive inclusions (FTLD-UPS) that stained negatively for tau, TDP-43, and FUS. Alzheimer's disease was uncommon (6%) among patients whose only top diagnosis during follow-up was bvFTD. Seventy-nine per cent of FTLD-tau, 86% of FTLD-TDP, and 88% of FTLD-FUS met at least 'possible' bvFTD diagnostic criteria at first presentation. The frequency of the six core bvFTD diagnostic features was similar in FTLD-tau and FTLD-TDP, suggesting that these features alone cannot be used to separate patients by major molecular class. Voxel-based morphometry revealed that nearly all pathological subgroups and even individual patients share atrophy in anterior cingulate, frontoinsula, striatum, and amygdala, indicating that degeneration of these regions is intimately linked to the behavioural syndrome produced by these diverse aetiologies. In addition to these unifying features, symptom profiles also differed among pathological subtypes, suggesting distinct anatomical vulnerabilities and informing a clinician's prediction of pathological diagnosis. Data-driven classification into one of the 10 most common pathological diagnoses was most accurate (up to 60.2%) when using a combination of known predictive factors (genetic mutations, motor features, or striking atrophy patterns) and the results of a discriminant function analysis that incorporated clinical, neuroimaging, and neuropsychological data.

Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia.

SEE SCABER AND TALBOT DOI101093/AWW264 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: A GGGGCC repeat expansion in C9orf72 leads to frontotemporal dementia and/or amyotrophic lateral sclerosis. Diverse pathological features have been identified, and their disease relevance remains much debated. Here, we describe two illuminating patients with frontotemporal dementia due to the C9orf72 repeat expansion. Case 1 was a 65-year-old female with behavioural variant frontotemporal dementia accompanied by focal degeneration in subgenual anterior cingulate cortex, amygdala, and medial pulvinar thalamus. At autopsy, widespread RNA foci and dipeptide repeat protein inclusions were observed, but TDP-43 pathology was nearly absent, even in degenerating brain regions. Case 2 was a 74-year-old female with atypical frontotemporal dementia-motor neuron disease who underwent temporal lobe resection for epilepsy 5 years prior to her first frontotemporal dementia symptoms. Archival surgical resection tissue contained RNA foci, dipeptide repeat protein inclusions, and loss of nuclear TDP-43 but no TDP-43 inclusions despite florid TDP-43 inclusions at autopsy 8 years after first symptoms. These findings suggest that C9orf72-specific phenomena may impact brain structure and function and emerge before first symptoms and TDP-43 aggregation.

Selective frontoinsular von Economo neuron and fork cell loss in early behavioral variant frontotemporal dementia.

Behavioral variant frontotemporal dementia (bvFTD) erodes complex social-emotional functions as the anterior cingulate cortex (ACC) and frontoinsula (FI) degenerate, but the early vulnerable neuron within these regions has remained uncertain. Previously, we demonstrated selective loss of ACC von Economo neurons (VENs) in bvFTD. Unlike ACC, FI contains a second conspicuous layer 5 neuronal morphotype, the fork cell, which has not been previously examined. Here, we investigated the selectivity, disease-specificity, laterality, timing, and symptom relevance of frontoinsular VEN and fork cell loss in bvFTD. Blinded, unbiased, systematic sampling was used to quantify bilateral FI VENs, fork cells, and neighboring neurons in 7 neurologically unaffected controls (NC), 5 patients with Alzheimer's disease (AD), and 9 patients with bvFTD, including 3 who died of comorbid motor neuron disease during very mild bvFTD. bvFTD showed selective FI VEN and fork cell loss compared with NC and AD, whereas in AD no significant VEN or fork cell loss was detected. Although VEN and fork cell losses in bvFTD were often asymmetric, no group-level hemispheric laterality effects were identified. Right-sided VEN and fork cell losses, however, correlated with each other and with anatomical, functional, and behavioral severity. This work identifies region-specific neuronal targets in early bvFTD.

Distinctive neurons of the anterior cingulate and frontoinsular cortex: a historical perspective.

Human anterior cingulate and frontoinsular cortices participate in healthy social-emotional processing. These regions feature 2 related layer 5 neuronal morphotypes, the von Economo neurons and fork cells. In this paper, we review the historical accounts of these neurons and provide a German-to-English translation of von Economo's seminal paper describing the neurons which have come to bear his name. We close with a brief discussion regarding the functional and clinical relevance of these neurons and their home regions.

IGFBP3 colocalizes with and regulates hypocretin (orexin).

BACKGROUND: The sleep disorder narcolepsy is caused by a vast reduction in neurons producing the hypocretin (orexin) neuropeptides. Based on the tight association with HLA, narcolepsy is believed to result from an autoimmune attack, but the cause of hypocretin cell loss is still unknown. We performed gene expression profiling in the hypothalamus to identify novel genes dysregulated in narcolepsy, as these may be the target of autoimmune attack or modulate hypocretin gene expression. METHODOLOGY/PRINCIPAL FINDINGS: We used microarrays to compare the transcriptome in the posterior hypothalamus of (1) narcoleptic versus control postmortem human brains and (2) transgenic mice lacking hypocretin neurons versus wild type mice. Hypocretin was the most downregulated gene in human narcolepsy brains. Among many additional candidates, only one, insulin-like growth factor binding protein 3 (IGFBP3), was downregulated in both human and mouse models and co-expressed in hypocretin neurons. Functional analysis indicated decreased hypocretin messenger RNA and peptide content, and increased sleep in transgenic mice overexpressing human IGFBP3, an effect possibly mediated through decreased hypocretin promotor activity in the presence of excessive IGFBP3. Although we found no IGFBP3 autoantibodies nor a genetic association with IGFBP3 polymorphisms in human narcolepsy, we found that an IGFBP3 polymorphism known to increase serum IGFBP3 levels was associated with lower CSF hypocretin-1 in normal individuals. CONCLUSIONS/SIGNIFICANCE: Comparison of the transcriptome in narcolepsy and narcolepsy model mouse brains revealed a novel dysregulated gene which colocalized in hypocretin cells. Functional analysis indicated that the identified IGFBP3 is a new regulator of hypocretin cell physiology that may be involved not only in the pathophysiology of narcolepsy, but also in the regulation of sleep in normal individuals, most notably during adolescence. Further studies are required to address the hypothesis that excessive IGFBP3 expression may initiate hypocretin cell death and cause narcolepsy.

Regulation of hypocretin (orexin) expression in embryonic zebrafish.

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.

Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms.

The suprachiasmatic nucleus (SCN) contains the brain's circadian pacemaker, but mechanisms by which it controls circadian rhythms of sleep and related behaviors are poorly understood. Previous anatomic evidence has implicated the dorsomedial hypothalamic nucleus (DMH) in circadian control of sleep, but this hypothesis remains untested. We now show that excitotoxic lesions of the DMH reduce circadian rhythms of wakefulness, feeding, locomotor activity, and serum corticosteroid levels by 78-89% while also reducing their overall daily levels. We also show that the DMH receives both direct and indirect SCN inputs and sends a mainly GABAergic projection to the sleep-promoting ventrolateral preoptic nucleus, and a mainly glutamate-thyrotropin-releasing hormone projection to the wake-promoting lateral hypothalamic area, including orexin (hypocretin) neurons. Through these pathways, the DMH may influence a wide range of behavioral circadian rhythms.

Selective activation of the extended ventrolateral preoptic nucleus during rapid eye movement sleep.

We found previously that damage to a cluster of sleep-active neurons (Fos-positive during sleep) in the ventrolateral preoptic nucleus (VLPO) decreases non-rapid eye movement (NREM) sleep in rats, whereas injury to the sleep-active cells extending dorsally and medially from the VLPO cluster (the extended VLPO) diminishes REM sleep. These results led us to examine whether neurons in the extended VLPO are activated during REM sleep and the connectivity of these neurons with pontine sites implicated in producing REM sleep: the laterodorsal tegmental nucleus (LDT), dorsal raphe nucleus (DRN), and locus ceruleus (LC). After periods of dark exposure that triggered enrichment of REM sleep, the number of Fos-positive cells in the extended VLPO was highly correlated with REM but not NREM sleep. In contrast, the number of Fos-positive cells in the VLPO cluster was correlated with NREM but not REM sleep. Sixty percent of sleep-active cells in the extended VLPO and 90% of sleep-active cells in the VLPO cluster in dark-treated animals contained galanin mRNA. Retrograde tracing from the LDT, DRN, and LC demonstrated more labeled cells in the extended VLPO than the VLPO cluster, and 50% of these in the extended VLPO were sleep-active. Anterograde tracing showed that projections from the extended VLPO and VLPO cluster targeted the cell bodies and dendrites of DRN serotoninergic neurons and LC noradrenergic neurons but were not apposed to cholinergic neurons in the LDT. The connections and physiological activity of the extended VLPO suggest a specialized role in the regulation of REM sleep.

Afferents to the ventrolateral preoptic nucleus.

Sleep is influenced by diverse factors such as circadian time, affective states, ambient temperature, pain, etc., but pathways mediating these influences are unknown. To identify pathways that may influence sleep, we examined afferents to the ventrolateral preoptic nucleus (VLPO), an area critically implicated in promoting sleep. Injections of the retrograde tracer cholera toxin B subunit (CTB) into the VLPO produced modest numbers of CTB-labeled monoaminergic neurons in the tuberomammillary nucleus, raphe nuclei, and ventrolateral medulla, as well as a few neurons in the locus coeruleus. Immunohistochemistry for monoaminergic markers showed dense innervation of the VLPO by histaminergic, noradrenergic, and serotonergic fibers. Along with previous findings, these results suggest that the VLPO and monoaminergic nuclei may be reciprocally connected. Retrograde and anterograde tracing showed moderate or heavy inputs to the VLPO from hypothalamic regions including the median preoptic nucleus, lateral hypothalamic area, and dorsomedial hypothalamic nucleus (DMH), autonomic regions including the infralimbic cortex and parabrachial nucleus, and limbic regions including the lateral septal nucleus and ventral subiculum. Light to moderate inputs arose from orexin and melanin concentrating hormone neurons, but cholinergic or dopaminergic inputs were extremely sparse. Suprachiasmatic nucleus (SCN) projections to the VLPO were sparse, but the heavy input to the VLPO from the DMH, which receives direct and indirect SCN inputs, could provide an alternate pathway regulating the circadian timing of sleep. These robust pathways suggest candidate mechanisms by which sleep may be influenced by brain systems regulating arousal, autonomic, limbic, and circadian functions.