VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction.

Alzheimer's disease (AD) patients exhibit neuropsychiatric symptoms that extend beyond classical cognitive deficits, suggesting involvement of subcortical areas. Here, we investigated the role of midbrain dopamine (DA) neurons in AD using the amyloid + tau-driven 3xTg-AD mouse model. We found deficits in reward-based operant learning in AD mice, suggesting possible VTA DA neuron dysregulation. Physiological assessment revealed hyperexcitability and disrupted firing in DA neurons caused by reduced activity of small-conductance calcium-activated potassium (SK) channels. RNA sequencing from contents of single patch-clamped DA neurons (Patch-seq) identified up-regulation of the SK channel modulator casein kinase 2 (CK2). Pharmacological inhibition of CK2 restored SK channel activity and normal firing patterns in 3xTg-AD mice. These findings shed light on a complex interplay between neuropsychiatric symptoms and subcortical circuits in AD, paving the way for novel treatment strategies.

Microglial MHC-I induction with aging and Alzheimer's is conserved in mouse models and humans.

Major histocompatibility complex I (MHC-I) CNS cellular localization and function is still being determined after previously being thought to be absent from the brain. MHC-I expression has been reported to increase with brain aging in mouse, rat, and human whole tissue analyses, but the cellular localization was undetermined. Neuronal MHC-I is proposed to regulate developmental synapse elimination and tau pathology in Alzheimer's disease (AD). Here, we report that across newly generated and publicly available ribosomal profiling, cell sorting, and single-cell data, microglia are the primary source of classical and non-classical MHC-I in mice and humans. Translating ribosome affinity purification-qPCR analysis of 3-6- and 18-22-month-old (m.o.) mice revealed significant age-related microglial induction of MHC-I pathway genes B2m, H2-D1, H2-K1, H2-M3, H2-Q6, and Tap1 but not in astrocytes and neurons. Across a timecourse (12-23 m.o.), microglial MHC-I gradually increased until 21 m.o. and then accelerated. MHC-I protein was enriched in microglia and increased with aging. Microglial expression, and absence in astrocytes and neurons, of MHC-I-binding leukocyte immunoglobulin-like (Lilrs) and paired immunoglobin-like type 2 (Pilrs) receptor families could enable cell -autonomous MHC-I signaling and increased with aging in mice and humans. Increased microglial MHC-I, Lilrs, and Pilrs were observed in multiple AD mouse models and human AD data across methods and studies. MHC-I expression correlated with p16INK4A, suggesting an association with cellular senescence. Conserved induction of MHC-I, Lilrs, and Pilrs with aging and AD opens the possibility of cell-autonomous MHC-I signaling to regulate microglial reactivation with aging and neurodegeneration.

Microglial MHC-I induction with aging and Alzheimer's is conserved in mouse models and humans.

Major Histocompatibility Complex I (MHC-I) CNS cellular localization and function is still being determined after previously being thought to be absent from the brain. MHC-I expression has been reported to increase with brain aging in mouse, rat, and human whole tissue analyses but the cellular localization was undetermined. Neuronal MHC-I is proposed to regulate developmental synapse elimination and tau pathology in Alzheimer's disease (AD). Here we report that across newly generated and publicly available ribosomal profiling, cell sorting, and single-cell data, microglia are the primary source of classical and non-classical MHC-I in mice and humans. Translating Ribosome Affinity Purification-qPCR analysis of 3-6 and 18-22 month old (m.o.) mice revealed significant age-related microglial induction of MHC-I pathway genes B2m , H2-D1 , H2-K1 , H2-M3 , H2-Q6 , and Tap1 but not in astrocytes and neurons. Across a timecourse (12-23 m.o.), microglial MHC-I gradually increased until 21 m.o. and then accelerated. MHC-I protein was enriched in microglia and increased with aging. Microglial expression, and absence in astrocytes and neurons, of MHC-I binding Leukocyte Immunoglobulin-like (Lilrs) and Paired immunoglobin-like type 2 (Pilrs) receptor families could enable cell-autonomous MHC-I signaling and increased with aging in mice and humans. Increased microglial MHC-I, Lilrs, and Pilrs were observed in multiple AD mouse models and human AD data across methods and studies. MHC-I expression correlated with p16INK4A , suggesting an association with cellular senescence. Conserved induction of MHC-I, Lilrs, and Pilrs with aging and AD opens the possibility of cell-autonomous MHC-I signaling to regulate microglial reactivation with aging and neurodegeneration.

Preservation of dendritic D2 receptor transmission in substantia nigra dopamine neurons with age.

Substantia nigra pars compacta (SNc) dopamine neurons are required for voluntary movement and reward learning, and advanced age is associated with motor and cognitive decline. In the midbrain, D2-type dopamine receptors located at dendrodendritic synapses between dopamine neurons control cell firing through G protein-activated potassium (GIRK) channels. We previously showed that aging disrupts dopamine neuron pacemaker firing in mice, but only in males. Here we show that the amplitude of D2-receptor inhibitory postsynaptic currents (D2-IPSCs) are moderately reduced in aged male mice. Local application of dopamine revealed a reduction in the amplitude of the D2-receptor currents in old males compared to young, pointing to a postsynaptic mechanism. Further experiments indicated that reduced D2 receptor signaling was not due to a general reduction in GIRK channel currents or degeneration of the dendritic arbor. Kinetic analysis showed no differences in D2-IPSC shape in old versus young mice or between sexes. Potentiation of D2-IPSCs by corticotropin releasing factor (CRF) was also not affected by age, indicating preservation of one mechanism of plasticity. These findings have implications for understanding dopamine transmission in aging, and reduced D2 receptor inhibition could contribute to increased susceptibility of males to SNc dopamine neuron degeneration in Parkinson's disease.

Neurotensin Release from Dopamine Neurons Drives Long-Term Depression of Substantia Nigra Dopamine Signaling.

Midbrain dopamine neurons play central physiological roles in voluntary movement, reward learning, and motivated behavior. Inhibitory signaling at somatodendritic dopamine D2 receptor (D2R) synapses modulates excitability of dopamine neurons. The neuropeptide neurotensin is expressed by many inputs to the midbrain and induces LTD of D2R synaptic currents (LTDDA); however, the source of neurotensin that is responsible for LTDDA is not known. Here we show, in brain slices from male and female mice, that LTDDA is driven by neurotensin released by dopamine neurons themselves. Optogenetic stimulation of dopamine neurons was sufficient to induce LTDDA in the substantia nigra, but not the VTA, and was dependent on neurotensin receptor signaling, postsynaptic calcium, and vacuolar-type H+-ATPase activity in the postsynaptic cell. These findings reveal a novel form of signaling between dopamine neurons involving release of the peptide neurotensin, which may act as a feedforward mechanism to increase dopamine neuron excitability.SIGNIFICANCE STATEMENT Dopamine neurons in the midbrain play a critical role in reward learning and the initiation of movement. Aberrant dopamine neuron function is implicated in a range of diseases and disorders, including Parkinson's disease, schizophrenia, obesity, and substance use disorders. D2 receptor-mediated PSCs are produced by a rare form of dendrodendritic synaptic transmission between dopamine neurons. These D2 receptor-mediated PSCs undergo LTD following application of the neuropeptide neurotensin. Here we show that release of neurotensin by dopamine neurons themselves is sufficient to induce LTD of dopamine transmission in the substantia nigra. Neurotensin signaling therefore mediates a second form of interdopamine neuron communication and may provide a mechanism by which dopamine neurons maintain excitability when nigral dopamine is elevated.

Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia.

Modern molecular and biochemical neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell type-specific events. This is particularly true in the analysis of minority glial populations in the brain, such as microglia, which may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer-based and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance. The Cx3cr1-NuTRAP mouse model was used to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile as a baseline comparison lacking cell isolation artifacts. All sorting methods performed similarly for microglial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the cell sorting. The cell preparation-induced activational phenotype could be minimized by inclusion of transcriptional and translational inhibitors or non-enzymatic dissociation conducted entirely at low temperatures. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts.

Repeated cocaine or methamphetamine treatment alters astrocytic CRF2 and GLAST expression in the ventral midbrain.

Dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) play a central role in the reinforcing properties of abused drugs including methamphetamine and cocaine. Chronic effects of psychostimulants in the SN/VTA also involve non-dopaminergic transmitters, including glutamate and the stress-related peptide corticotropin-releasing factor (CRF). In the SN/VTA, astrocytes express a variety of membrane-bound neurotransmitter receptors and transporters that influence neurotransmission. CRF receptor type 2 (CRF2) activity in the VTA is important for stress-induced relapse and drug-seeking behaviour, but the localization of its effects is incompletely understood. Here, we first identified CRF2 transcript in astrocytes of the SN/VTA using RNA-Seq in Aldh1l1;NuTRAP mice and confirmed it using in situ hybridization (RNAscope) in wild-type mice. We then used immunofluorescence to quantify the astrocytic marker protein S100ß, glial-specific glutamate/aspartate transporter GLAST, and CRF2 in the SN/VTA following 12 days of treatment (i.p.) with methamphetamine (3 mg/kg), cocaine (10 mg/kg), or saline. We observed a significant decrease in GLAST immunofluorescence in brains of psychostimulant treated mice compared with saline controls. In addition, we observed increased labelling of CRF2 in drug treated groups, a decrease in the number of S100ß positive cells, and an increase of co-staining of CRF2 with both S100ß and tyrosine hydroxylase (dopamine neurons). Our results suggest a significant interaction between CRF2, GLAST, and astrocytes in the midbrain that emerges with repeated exposure to psychostimulants. These findings provide rationale for future investigation of astrocyte-based strategies for altering cellular and circuit function in response to stress and drug exposure.