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.

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.

Neurotensin receptor 1 deletion decreases methamphetamine self-administration and the associated reduction in dopamine cell firing.

We previously reported that a non-selective pharmacological blockade of neurotensin receptors in the ventral tegmental area (VTA) decreases methamphetamine (METH) self-administration in mice. Here, we explored the consequences of genetic deletion of neurotensin receptor 1 (NtsR1) on METH self-administration and VTA dopamine neuron firing activity. We implanted mice with an indwelling jugular catheter and trained them to nose-poke for intravenous infusions of METH. Mice with NtsR1 deletion (KO) acquired self-administration similar to wildtype (WT) and heterozygous (HET) littermates. However, in NtsR1 KO and HET mice, METH intake and motivated METH seeking decreased when the response requirement was increased to a fixed ratio 3 and when mice were tested on a progressive ratio protocol. After completion of METH self-administration, single cell in vivo extracellular recordings of dopamine firing activity in the VTA were obtained in anesthetized mice. Non-bursting dopamine neurons from KO mice fired at slower rates than those from WT mice, supporting an excitatory role for NtsR1 on VTA dopamine neuronal activity. In WT mice, a history of METH self-administration decreased dopamine cell firing frequency compared with cells from drug-naïve controls. NtsR1 KO and HET mice did not exhibit this decline in dopamine cell firing activity after METH experience. We also observed an increase in population activity following METH self-administration that was strongest in the WT group. Our results suggest a role for NtsR1 in METH-seeking behavior and indicate that ablation of NtsR1 prevents the detrimental effects of prolonged METH self-administration on VTA dopamine cell firing frequency.

A history of ethanol drinking increases locomotor stimulation and blunts enhancement of dendritic dopamine transmission by methamphetamine.

Ethanol and psychostimulant use disorders exhibit comorbidity in humans and cross-sensitization in animal models, but the neurobiological underpinnings of this are not well understood. Ethanol acutely increases dopamine neuron excitability, and psychostimulants such as cocaine or methamphetamine increase extracellular dopamine through inhibition of uptake through the dopamine transporter (DAT) and/or vesicular monoamine transporter 2 (VMAT2). Psychostimulants also depress dopamine neuron activity by enhancing dendritic dopamine neurotransmission. Here, we show that mice with a previous history of ethanol drinking are more sensitive to the locomotor-stimulating effects of a high dose (5 mg/kg), but not lower doses (1 and 3 mg/kg) of methamphetamine or any tested dose of cocaine (3, 10, and 18 mg/kg), compared with water-drinking controls. We next investigated the impact of a history of ethanol drinking, in a separate group of mice, on methamphetamine- or cocaine-induced enhancement of dendritic dopamine transmission using whole-cell voltage clamp electrophysiology in mouse brain slices. Methamphetamine, applied at a concentration (10 µM) that affects both DAT and VMAT2, enhanced D2 receptor-mediated inhibitory postsynaptic currents (D2-IPSCs) in both groups, but this effect was blunted in mice with a history of ethanol drinking. As methamphetamine action at VMAT2 disrupts dopamine neurotransmission, these results may suggest enhanced action of methamphetamine at VMAT2. Furthermore, there were no differences in low-dose methamphetamine or cocaine-induced enhancement of D2-IPSCs, suggesting intact DAT function. Disruption of methamphetamine-induced enhancement of dendritic dopamine transmission would result in decreased inhibition of dopamine neurons, ultimately increasing downstream release and the behavioral effects of methamphetamine.

Diverse actions of the modulatory peptide neurotensin on central synaptic transmission.

Neurotensin (NT) is a 13 amino acid neuropeptide that is expressed throughout the central nervous system and is implicated in the etiology of multiple diseases and disorders. Many primary investigations of NT-induced modulation of neuronal excitability at the level of the synapse have been conducted, but they have not been summarized in review form in nearly 30 years. Therefore, the goal of this review is to discuss the many actions of NT on neuronal excitability across brain regions as well as NT circuit architecture. In the basal ganglia as well as other brain nuclei, NT can act through diverse intracellular signaling cascades to enhance or depress neuronal activity by modulating activity of ion channels, ionotropic and metabotropic neurotransmitter receptors, and presynaptic release of neurotransmitters. Further, NT can produce indirect effects by evoking endocannabinoid release, and recently has itself been identified as a putative retrograde messenger. In the basal ganglia, the diverse actions and circuit architecture of NT signaling allow for input-specific control of reward-related behaviors.

Antagonism of Neurotensin Receptors in the Ventral Tegmental Area Decreases Methamphetamine Self-Administration and Methamphetamine Seeking in Mice.

Background: Neurotensin is a peptide that modulates central dopamine neurotransmission and dopamine-related behaviors. Methamphetamine self-administration increases neurotensin levels in the ventral tegmental area, but the consequences for self-administration behavior have not been described. Here we test the hypothesis that antagonizing neurotensin receptors in the ventral tegmental area attenuates the acquisition of methamphetamine self-administration and methamphetamine intake. Methods: We implanted mice with an indwelling catheter in the right jugular vein and bilateral cannulae directed at the ventral tegmental area. Mice were then trained to nose-poke for i.v. infusions of methamphetamine (0.1 mg/kg/infusion) on a fixed ratio 3 schedule. Results: Mice receiving microinfusions of the neurotensin NTS1/NTS2 receptor antagonist SR142948A in the ventral tegmental area (10 ng/side) prior to the first 5 days of methamphetamine self-administration required more sessions to reach acquisition criteria. Methamphetamine intake was decreased in SR142948A-treated mice both during training and later during maintenance of self-administration. Drug seeking during extinction, cue-induced reinstatement, and progressive ratio schedules was also reduced in the SR142948A group. The effects of SR142948A were not related to changes in basal locomotor activity or methamphetamine psychomotor properties. In both SR142948A- and saline-treated mice, a strong positive correlation between methamphetamine intake and enhanced locomotor activity was observed. Conclusion: Our results suggest that neurotensin input in the ventral tegmental area during initial methamphetamine exposure contributes to the acquisition of methamphetamine self-administration and modulates later intake and methamphetamine-seeking behavior in mice. Furthermore, our results highlight the role of endogenous neurotensin in the ventral tegmental area in the reinforcing efficacy of methamphetamine, independent of its psychomotor effects.

Progressively disrupted somatodendritic morphology in dopamine neurons in a mouse Parkinson's model.

BACKGROUND: Parkinson's disease is characterized by the progressive loss of dopamine neurons in the substantia nigra, leading to severe motor deficits. Although the disease likely begins to develop years before observable motor symptoms, the specific morphological and functional alterations involved are poorly understood. OBJECTIVES: MitoPark mice lack the gene coding for mitochondrial transcription factor A specifically in dopamine neurons, which over time produces a progressive decline of neuronal function and related behavior that phenotypically mirrors human parkinsonism. Our previous work identified a progressive decrease in cell capacitance in dopamine neurons from MitoPark mice, possibly suggesting reduced membrane surface area. We therefore sought to identify and quantify somatodendritic parameters in this model across age. METHODS: We used whole-cell patch clamp and fluorescent labeling to quantify somatodendritic morphology of single, neurobiotin-filled dopamine neurons in acutely isolated brain slices from MitoPark mice. RESULTS: We found that MitoPark mice exhibit an adult-onset, age-dependent reduction of neuritic branching and soma size in dopamine neurons. This decline proceeds similarly in MitoPark mice of both sexes, but does not begin until after the age that early decrements in ion channel physiology and behavior have previously been observed. CONCLUSIONS: A progressive and severe decline in somatodendritic morphology occurs prior to cell death, but is not responsible for the subtle decrements observable in the earliest stages of neurodegeneration. This work could help identify the ideal time window for specific treatments to halt disease progression and avert debilitating motor deficits in Parkinson's patients. © 2018 International Parkinson and Movement Disorder Society.

Dopaminergic Neurons Exhibit an Age-Dependent Decline in Electrophysiological Parameters in the MitoPark Mouse Model of Parkinson's Disease.

Dopaminergic neurons of the substantia nigra (SN) play a vital role in everyday tasks, such as reward-related behavior and voluntary movement, and excessive loss of these neurons is a primary hallmark of Parkinson's disease (PD). Mitochondrial dysfunction has long been implicated in PD and many animal models induce parkinsonian features by disrupting mitochondrial function. MitoPark mice are a recently developed genetic model of PD that lacks the gene for mitochondrial transcription factor A specifically in dopaminergic neurons. This model mimics many distinct characteristics of PD including progressive and selective loss of SN dopamine neurons, motor deficits that are improved byl-DOPA, and development of inclusion bodies. Here, we used brain slice electrophysiology to construct a timeline of functional decline in SN dopaminergic neurons from MitoPark mice. Dopaminergic neurons from MitoPark mice exhibited decreased cell capacitance and increased input resistance that became more severe with age. Pacemaker firing regularity was disrupted in MitoPark mice and ion channel conductances associated with firing were decreased. Additionally, dopaminergic neurons from MitoPark mice showed a progressive decrease of endogenous dopamine levels, decreased dopamine release, and smaller D2 dopamine receptor-mediated outward currents. Interestingly, expression of ion channel subunits associated with impulse activity (Cav1.2, Cav1.3, HCN1, Nav1.2, and NavB3) was upregulated in older MitoPark mice. The results describe alterations in intrinsic and synaptic properties of dopaminergic neurons in MitoPark mice occurring at ages both before and concurrent with motor impairment. These findings may help inform future investigations into treatment targets for prodromal PD. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) is the second most diagnosed neurodegenerative disorder, and the classic motor symptoms of the disease are attributed to selective loss of dopaminergic neurons of the substantia nigra. The MitoPark mouse is a genetic model of PD that mimics many of the key characteristics of the disease and enables the study of progressive neurodegeneration in parkinsonism. Here we have identified functional deficits in the ion channel physiology of dopaminergic neurons from MitoPark mice that both precede and are concurrent with the time course of behavioral symptomatology. Because PD is a progressive disease with a long asymptomatic phase, identification of early functional adaptations could lay the groundwork to test therapeutic interventions that halt or reverse disease progression.

Neurotensin Induces Presynaptic Depression of D2 Dopamine Autoreceptor-Mediated Neurotransmission in Midbrain Dopaminergic Neurons.

Increased dopaminergic signaling is a hallmark of severe mesencephalic pathologies such as schizophrenia and psychostimulant abuse. Activity of midbrain dopaminergic neurons is under strict control of inhibitory D2 autoreceptors. Application of the modulatory peptide neurotensin (NT) to midbrain dopaminergic neurons transiently increases activity by decreasing D2 dopamine autoreceptor function, yet little is known about the mechanisms that underlie long-lasting effects. Here, we performed patch-clamp electrophysiology and fast-scan cyclic voltammetry in mouse brain slices to determine the effects of NT on dopamine autoreceptor-mediated neurotransmission. Application of the active peptide fragment NT8-13 produced synaptic depression that exhibited short- and long-term components. Sustained depression of D2 autoreceptor signaling required activation of the type 2 NT receptor and the protein phosphatase calcineurin. NT application increased paired-pulse ratios and decreased extracellular levels of somatodendritic dopamine, consistent with a decrease in presynaptic dopamine release. Surprisingly, we observed that electrically induced long-term depression of dopaminergic neurotransmission that we reported previously was also dependent on type 2 NT receptors and calcineurin. Because electrically induced depression, but not NT-induced depression, was blocked by postsynaptic calcium chelation, our findings suggest that endogenous NT may act through a local circuit to decrease presynaptic dopamine release. The current research provides a mechanism through which augmented NT release can produce a long-lasting increase in membrane excitability of midbrain dopamine neurons. SIGNIFICANCE STATEMENT: Whereas plasticity of glutamate synapses in the brain has been studied extensively, demonstrations of plasticity at dopaminergic synapses have been more elusive. By quantifying inhibitory neurotransmission between midbrain dopaminergic neurons in brain slices from mice we have discovered that the modulatory peptide neurotensin can induce a persistent synaptic depression by decreasing dopamine release. This depression of inhibitory synaptic input would be expected to increase excitability of dopaminergic neurons. Induction of the plasticity can be pharmacologically blocked by antagonists of either the protein phosphatase calcineurin or neurotensin receptors, and persists surprisingly long after a brief exposure to the peptide. Since neurotensin-dopamine interactions have been implicated in hyperdopaminergic pathologies, these findings describe a synaptic mechanism that could contribute to addiction and/or schizophrenia.

Aging decreases L-type calcium channel currents and pacemaker firing fidelity in substantia nigra dopamine neurons.

Substantia nigra dopamine neurons are involved in behavioral processes that include cognition, reward learning, and voluntary movement. Selective deterioration of these neurons is responsible for the motor deficits associated with Parkinson's disease (PD). Aging is the leading risk factor for PD, suggesting that adaptations occurring in dopamine neurons during normal aging may predispose individuals to the development of PD. Previous studies suggest that the unique set of ion conductances that drive spontaneous, rhythmic firing of action potentials could predispose substantia nigra dopamine neurons to selective neurodegeneration. Here we show, using patch-clamp electrophysiological recordings in brain slices, that substantia nigra dopamine neurons from mice 25-30 months of age (old) have comparable membrane capacitance and input resistance to neurons from mice 2-7 months of age (young). However, neurons from old mice exhibit slower firing rates, narrower spike widths, and more variable interspike intervals compared with neurons from young mice. Dopamine neurons from old mice also exhibit smaller L-type calcium channel currents, providing a plausible mechanism that likely contributes to the changes in impulse activity. Age-related decrements in the physiological function of dopamine neurons could contribute to the decrease in voluntary movement and other dopamine-mediated behaviors observed in aging populations. Furthermore, as pharmacological antagonism of L-type calcium channels has been proposed as a potential treatment for the early stages of PD, our results could point to a limited temporal window of opportunity for this therapeutic intervention.

Food restriction increases glutamate receptor-mediated burst firing of dopamine neurons.

Restriction of food intake increases the acquisition of drug abuse behavior and enhances the reinforcing efficacy of those drugs. However, the neurophysiological mechanisms responsible for the interactions between feeding state and drug use are largely unknown. Here we show that chronic mild food restriction increases the burst firing of dopamine neurons in the substantia nigra. Dopamine neurons from food-restricted mice exhibited increased burst firing in vivo, an effect that was enhanced by an injection of the psychomotor stimulant cocaine (10 mg/kg, i.p.). Food restriction also enhanced aspartic acid-induced burst firing of dopamine neurons in an ex vivo brain slice preparation, consistent with an adaptation occurring in the somatodendritic compartment and independent of a circuit mechanism. Enhanced burst firing persisted after 10 d of free feeding following chronic food restriction but was not observed following a single overnight fast. Whole-cell patch-clamp recordings indicated that food restriction also increased electrically evoked AMPAR/NMDAR ratios and increased D2 autoreceptor-mediated desensitization in dopamine neurons. These results identify dopamine neurons in the substantia nigra as a convergence point for the interactions between feeding state and drugs of abuse. Furthermore, increased glutamate transmission combined with decreased autoreceptor inhibition could work in concert to enhance drug efficacy in response to food restriction.

Methamphetamine self-administration in mice decreases GIRK channel-mediated currents in midbrain dopamine neurons.

BACKGROUND: Methamphetamine is a psychomotor stimulant with abuse liability and a substrate for catecholamine uptake transporters. Acute methamphetamine elevates extracellular dopamine, which in the midbrain can activate D2 autoreceptors to increase a G-protein gated inwardly rectifying potassium (GIRK) conductance that inhibits dopamine neuron firing. These studies examined the neurophysiological consequences of methamphetamine self-administration on GIRK channel-mediated currents in dopaminergic neurons in the substantia nigra and ventral tegmental area. METHODS: Male DBA/2J mice were trained to self-administer intravenous methamphetamine. A dose response was conducted as well as extinction and cue-induced reinstatement. In a second study, after at least 2 weeks of stable self-administration of methamphetamine, electrophysiological brain slice recordings were conducted on dopamine neurons from self-administering and control mice. RESULTS: In the first experiment, ad libitum-fed, nonfood-trained mice exhibited a significant increase in intake and locomotion following self-administration as the concentration of methamphetamine per infusion was increased (0.0015-0.15mg/kg/infusion). Mice exhibited extinction in responding and cue-induced reinstatement. In the second experiment, dopamine cells in both the substantia nigra and ventral tegmental area from adult mice with a history of methamphetamine self-administration exhibited significantly smaller D2 and GABAB receptor-mediated currents compared with control mice, regardless of whether their daily self-administration sessions had been 1 or 4 hours. Interestingly, the effects of methamphetamine self-administration were not present when intracellular calcium was chelated by including BAPTA in the recording pipette. CONCLUSIONS: Our results suggest that methamphetamine self-administration decreases GIRK channel-mediated currents in dopaminergic neurons and that this effect may be calcium dependent.

SK and Kv4 Channels Limit Spike Timing Perturbations in Pacemaking Dopamine Neurons.

Midbrain dopamine (DA) neurons are among the best characterized pacemaker neurons, having intrinsic, rhythmic firing activity even in the absence of synaptic input. However, the mechanisms of DA neuron pacemaking have not been systematically related to how these cells respond to synaptic input. The input-output properties of pacemaking neurons can be characterized by the phase-resetting curve (PRC), which describes the sensitivity of interspike interval (ISI) length to inputs arriving at different phases of the firing cycle. Here we determined PRCs of putative DA neurons in the substantia nigra pars compacta in brain slices from male and female mice using gramicidin-perforated current-clamp recordings with electrical noise stimuli applied through the patch pipette. On average, and compared with nearby putative GABA neurons, DA neurons showed a low, nearly constant level of sensitivity across most of the ISI, but individual cells had PRCs showing relatively greater sensitivity at early or late phases. Pharmacological experiments showed that DA neuron PRCs are shaped by small-conductance calcium-activated potassium and Kv4 channels, which limit input sensitivity across early and late phases of the ISI. Our results establish the PRC as a tractable experimental measurement of individual DA neuron input-output relationships and identify two of the major ionic conductances that limit perturbations to rhythmic firing. These findings have applications in modeling and for identifying biophysical changes in response to disease or environmental manipulations.

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.

Long-term methamphetamine self-administration increases mesolimbic mitochondrial oxygen consumption and decreases striatal glutathione.

Neurotoxic regimens of methamphetamine (METH) are known to increase reactive oxygen species (ROS), affect redox homeostasis, and lead to damage in dopamine neurons. Functional changes induced by long-term METH self-administration on mitochondrial respiratory metabolism and redox homeostasis are less known. To fill this gap, we implanted a jugular catheter into adult male mice and trained them to nose poke for METH infusions. After several weeks of METH exposure, we collected samples of the ventral striatum (vST) and the ventral midbrain (vMB). We used HPLC to determine the levels of the ROS scavenger glutathione in its reduced (GSH) and oxidized forms. Then, we used high-resolution respirometry to determine the oxygen consumption rate (OCR) of mitochondrial complexes. Finally, using in vivo electrophysiology, we assessed changes in dopamine neuron firing activity in the VTA. METH self-administration produced a decrease of the GSH pool in vST, correlating with lifetime METH intake. We observed increased mitochondrial respiration across the two mesolimbic regions. METH self-administration decreases firing rate and burst activity but increases the number of spontaneously active dopamine neurons per track. We conclude that METH self-administration progressively decreased the antioxidant pool in sites of higher dopamine release and produced an increase in mitochondrial metabolism in the mesolimbic areas, probably derived from the increased number of dopamine neurons actively firing. However, dopamine neuron firing activity is decreased by METH self-administration, reflecting a new basal level of dopamine neurotransmission.

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.

Specificity and efficiency of tamoxifen-mediated Cre induction is equivalent regardless of age.

Temporally controlling Cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report early post-natal/juvenile (<2 m.o.) Tam induction, but age-related neurodegeneration and aging studies can require Cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam-mediated Cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare Cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at early and late ages. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each Cre and flox mouse line should be independently validated, however, these findings demonstrate that Tam-mediated Cre induction can be performed even into older mouse ages and should be generalizable to other inducible Cre models.

Consistent specificity and efficiency of tamoxifen-mediated cre induction across ages.

Temporally controlling cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report Tam induction at early post-natal/juvenile (<2 m.o.) mouse ages, but age-related neurodegeneration and aging studies can require cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam mediated cre induction at early and late ages. Here, microglial-specific Cx3cr1 creERT 2 mice were crossed to a floxed NuTRAP reporter to compare cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at 3-6 m.o. or 20 m.o. of age. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each cre and flox mouse line should be validated independently, however, these findings demonstrate that Tam-mediated cre induction can be performed even into older mouse ages.

Neuronal deletion of MnSOD in mice leads to demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Neuronal oxidative stress has been implicated in aging and neurodegenerative disease. Here we investigated the impact of elevated oxidative stress induced in mouse spinal cord by deletion of Mn-Superoxide dismutase (MnSOD) using a neuron specific Cre recombinase in Sod2 floxed mice (i-mn-Sod2 KO). Sod2 deletion in spinal cord neurons was associated with mitochondrial alterations and peroxide generation. Phenotypically, i-mn-Sod2 KO mice experienced hindlimb paralysis and clasping behavior associated with extensive demyelination and reduced nerve conduction velocity, axonal degeneration, enhanced blood brain barrier permeability, elevated inflammatory cytokines, microglia activation, infiltration of neutrophils and necroptosis in spinal cord. In contrast, spinal cord motor neuron number, innervation of neuromuscular junctions, muscle mass, and contractile function were not altered. Overall, our findings show that loss of MnSOD in spinal cord promotes a phenotype of demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

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.