A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease.

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

Neuroanatomical and cognitive biomarkers of alpha-synuclein propagation in a mouse model of synucleinopathy prior to onset of motor symptoms.

Significant evidence suggests that misfolded alpha-synuclein (aSyn), a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinson's disease (PD) and other synucleinopathies. In fact, timed inoculation of M83 hemizygous mice with recombinant human aSyn preformed fibrils (PFF) has shown symptomatic deficits after substantial spreading of pathogenic alpha-synuclein, as detected by markers for the phosphorylation of S129 of aSyn. However, whether accumulated toxicity impact human-relevant cognitive and structural neuroanatomical measures is not fully understood. Here we performed a single unilateral striatal PFF injection in M83 hemizygous mice, and using two assays with translational potential, ex vivo magnetic resonance imaging (MRI) and touchscreen testing, we examined the combined neuroanatomical and behavioral impact of aSyn propagation. In PFF-injected mice, we observed widespread atrophy in bilateral regions that project to or receive input from the injection site using MRI. We also identified early deficits in reversal learning prior to the emergence of motor symptoms. Our findings highlight a network of regions with related cellular correlates of pathology that follow the progression of aSyn spreading, and that affect brain areas relevant for reversal learning. Our experiments suggest that M83 hemizygous mice injected with human PFF provides a model to understand how misfolded aSyn affects human-relevant pre-clinical measures and suggest that these pre-clinical biomarkers could be used to detect early toxicity of aSyn and provide better translational measures between mice and human disease.

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo.

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.

Hypocretin1/orexinA-immunoreactive axons form few synaptic contacts on rat ventral tegmental area neurons that project to the medial prefrontal cortex.

BACKGROUND: Hypocretins/orexins (Hcrt/Ox) are hypothalamic neuropeptides involved in sleep-wakefulness regulation. Deficiency in Hcrt/Ox neurotransmission results in the sleep disorder narcolepsy, which is characterized by an inability to maintain wakefulness. The Hcrt/Ox neurons are maximally active during wakefulness and project widely to the ventral tegmental area (VTA). A dopamine-containing nucleus projecting extensively to the cerebral cortex, the VTA enhances wakefulness. In the present study, we used retrograde tracing from the medial prefrontal cortex (mPFC) to examine whether Hcrt1/OxA neurons target VTA neurons that could sustain behavioral wakefulness through their projections to mPFC. RESULTS: The retrograde tracer Fluorogold (FG) was injected into mPFC and, after an optimal survival period, sections through the VTA were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Most VTA neurons projecting to the mPFC were located in the parabrachial nucleus of the ipsilateral VTA and were non-dopaminergic. Only axonal profiles showed Hcrt1/OxA-immunoreactivity in VTA. Hcrt1/OxA reactivity was observed in axonal boutons and many unmyelinated axons. The Hcrt1/OxA immunoreactivity was found filling axons but it was also observed in parts of the cytoplasm and dense-core vesicles. Hcrt1/OxA-labeled boutons frequently apposed FG-immunolabeled dendrites. However, Hcrt1/OxA-labeled boutons rarely established synapses, which, when they were established, were mainly asymmetric (excitatory-type), with either FG-labeled or unlabeled dendrites. CONCLUSIONS: Our results provide ultrastructural evidence that Hcrt1/OxA neurons may exert a direct synaptic influence on mesocortical neurons that would facilitate arousal and wakefulness. The paucity of synapses, however, suggest that the activity of VTA neurons with cortical projections might also be modulated by Hcrt1/OxA non-synaptic actions. In addition, Hcrt1/OxA could modulate the postsynaptic excitatory responses of VTA neurons with cortical projections to a co-released excitatory transmitter from Hcrt1/OxA axons. Our observation of Hcrt1/OxA targeting of mesocortical neurons supports Hcrt1/OxA wakefulness enhancement in the VTA and could help explain the characteristic hypersomnia present in narcoleptic patients.

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.

Background: Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Methods: Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. Results: We first identified specific non-coding variants in CTSB that drive the association with PD and are linked to changes in brain CTSB expression levels. Using iPSC-derived dopaminergic neurons we then find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. Moreover, in cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. Similarly, in midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition or knockout potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. Conclusions: The results of our genetic and functional studies indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy.

There is conflicting evidence regarding the mechanisms of α-synuclein internalization, and its trafficking itinerary following cellular entry remains largely unknown. To examine these issues, we describe steps for coupling α-synuclein preformed fibrils (PFFs) to nanogold beads and their subsequent characterization by electron microscopy (EM). Then we describe the uptake of conjugated PFFs by U2OS cells plated on Permanox 8-well chamber slides. This process eliminates the reliance on antibody specificity and the need to employ complex immunoEM staining protocols. For complete details on the use and execution of this protocol, please refer to Bayati et al. (2022).1.

USP19 deubiquitinase inactivation regulates α-synuclein ubiquitination and inhibits accumulation of Lewy body-like aggregates in mice.

The USP19 deubiquitinase is found in a locus associated with Parkinson's Disease (PD), interacts with chaperonins, and promotes secretion of α-synuclein (α-syn) through the misfolding-associated protein secretion (MAPS) pathway. Since these processes might modulate the processing of α-syn aggregates in PD, we inactivated USP19 (KO) in mice expressing the A53T mutation of α-syn and in whom α-syn preformed fibrils (PFF) had been injected in the striatum. Compared to WT, KO brains showed decreased accumulation of phospho-synuclein (pSyn) positive aggregates. This improvement was associated with less activation of microglia and improved performance in a tail-suspension test. Exposure of primary neurons from WT and KO mice to PFF in vitro also led to decreased accumulation of pSyn aggregates. KO did not affect uptake of PFF nor propagation of aggregates in the cultured neurons. We conclude that USP19 instead modulates intracellular dynamics of aggregates. At an early time following PFF injection when the number of pSyn-positive neurons were similar in WT and KO brains, the KO neurons contained less aggregates. KO brain aggregates stained more intensely with anti-ubiquitin antibodies. Immunoprecipitation of soluble proteins from WT and KO brains with antibodies to pSyn showed higher levels of ubiquitinated oligomeric species in the KO samples. We propose that the improved pathology in USP19 KO brains may arise from decreased formation or enhanced clearance of the more ubiquitinated aggregates and/or enhanced disassembly towards more soluble oligomeric species. USP19 inhibition may represent a novel therapeutic approach that targets the intracellular dynamics of α-syn complexes.

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Somatostatin varicosities contain the vesicular GABA transporter and contact orexin neurons in the hypothalamus.

Somatostatin (SST) is a neuropeptide with known inhibitory actions in the hypothalamus, where it inhibits release of growth hormone-releasing hormone (GHRH), while also influencing the sleep-wake cycle. Here we investigated in the rat whether SST neurons might additionally release GABA (gamma-aminobutyric acid) or glutamate in different regions and whether they might contact orexin neurons that play an important role in the maintenance of wakefulness. In dual-immunostained sections viewed by epifluorescence microscopy, we examined if SST varicosities were immunopositive for the vesicular transporter for GABA (VGAT) or glutamate (VGLUT2) in the posterolateral hypothalamus and neighboring arcuate nucleus and median eminence. Of the SST varicosities in the posterolateral hypothalamus, 18% were immunopositive for VGAT, whereas ≤ 1% were immunopositive for VGLUT2. In the arcuate and median eminence, 26 and 64% were VGAT+ and < 3% VGLUT2 + , respectively. In triple-immunostained sections viewed by epifluorescence and confocal microscopy, SST varicosities were seen in contact with orexin somata, and of these varicosities, a significant proportion (23%) contained VGAT along with synaptophysin, the presynaptic marker for small synaptic vesicles, and a similar proportion (25%) abutted puncta that were immunostained for gephyrin, the postsynaptic marker for GABAergic synapses. Our results indicate that a significant proportion of SST varicosities in the hypothalamus have the capacity to release GABA, to form inhibitory synapses upon orexin neurons, and accordingly through their peptide and/or amino acid, to inhibit orexin neurons, as well as GHRH neurons. Thus while regulating GHRH release, SST neurons could serve to attenuate arousal and permit progression through the sleep cycle.

Rapid macropinocytic transfer of α-synuclein to lysosomes.

The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinson's disease (PD) and other synucleinopathies; however, the mechanisms underlying internalization and cellular spread are enigmatic. Here, we use confocal and superresolution microscopy, subcellular fractionation, and electron microscopy (EM) of immunogold-labeled α-synuclein preformed fibrils (PFFs) to demonstrate that this form of the protein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2 min. Uptake of PFFs is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labeled PFFs are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFFs use a unique internalization mechanism as a component of cell-to-cell propagation.

Co-registration of Imaging Modalities (MRI, CT and PET) to Perform Frameless Stereotaxic Robotic Injections in the Common Marmoset.

The common marmoset has emerged as a popular model in neuroscience research, in part due to its reproductive efficiency, genetic and neuroanatomical similarities to humans and the successful generation of transgenic lines. Stereotaxic procedures in marmosets are guided by 2D stereotaxic atlases, which are constructed with a limited number of animals and fail to account for inter-individual variability in skull and brain size. Here, we developed a frameless imaging-guided stereotaxic system that improves upon traditional approaches by using subject-specific registration of computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) data to identify a surgical target, namely the putamen, in two marmosets. The skull surface was laser-scanned to create a point cloud that was registered to the 3D reconstruction of the skull from CT. Reconstruction of the skull, as well as of the brain from MR images, was crucial for surgical planning. Localisation and injection into the putamen was done using a 6-axis robotic arm controlled by a surgical navigation software (Brainsight™). Integration of subject-specific registration and frameless stereotaxic navigation allowed target localisation specific to each animal. Injection of alpha-synuclein fibrils into the putamen triggered progressive neurodegeneration of the nigro-striatal system, a key feature of Parkinson's disease. Four months post-surgery, a PET scan found evidence of nigro-striatal denervation, supporting accurate targeting of the putamen during co-registration and subsequent surgery. Our results suggest that this approach, coupled with frameless stereotaxic neuronavigation, is accurate in localising surgical targets and can be used to assess endpoints for longitudinal studies.

Hypocretin1/OrexinA-containing axons innervate locus coeruleus neurons that project to the Rat medial prefrontal cortex. Implication in the sleep-wakefulness cycle and cortical activation.

The Hypocretin1/OrexinA (Hcrt1/OxA) neuropeptides are found in a group of posterolateral hypothalamus neurons and are involved in sleep-wakefulness cycle regulation. Hcrt1/OxA neurons project widely to brainstem aminergic structures, such as the locus coeruleus (LC), which are involved in maintenance of wakefulness and EEG activation through intense projections to the medial prefrontal cortex (mPFC). Moreover, defects of the Hcrt1/OxA system are linked to narcolepsy, a disorder characterized by excessive diurnal hypersomnia and REM state disturbance. We aimed to determine whether Hcrt1/OxA neurons innervate LC neurons (noradrenergic and nonnoradrenergic) that project to the mPFC, thereby sustaining behavioral wakefulness. To assess this, we used retrograde tracing from mPFC injections and either Hcrt1/OxA or tyrosine hydroxylase (TH) immunohistochemical labeling in single sections of rat LC. The retrograde tracer Fluorogold (FG) was microinjected into mPFC and, at optimal survival periods, sections through the LC were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Many LC neurons projecting to mPFC were nonnoradrenergic. Electron microscopy revealed a prominent localization of Hcrt1/OxA in unmyelinated axons and axon boutons (varicosities and axon terminals) within the LC. Hcrt1/OxA-immunoreactive axon boutons frequently apposed (104/1907) or made asymmetric excitatory-type synapses (60/1907) with FG-immunolabeled dendrites, indicating that Hcrt1/OxA can modulate the activity of LC neurons with cortical projections. Our results show that Hcrt1/OxA hypothalamic neurons likely excite LC neurons that project to the mPFC, and thus activate EEG and facilitate wakefulness. In narcoleptics, who are deficient in Hcrt1/OxA, impairment of this Hcrt1/OxA hypothalamic input to LC might contribute to the appearance of excessive daytime sleepiness.

Pharmacological Inhibition of Brain EGFR Activation By a BBB-penetrating Inhibitor, AZD3759, Attenuates α-synuclein Pathology in a Mouse Model of α-Synuclein Propagation.

Aggregation and deposition of α-synuclein (α-syn) in Lewy bodies within dopamine neurons of substantia nigra (SN) is the pathological hallmark of Parkinson's disease (PD). These toxic α-syn aggregates are believed to propagate from neuron-to-neuron and spread the α-syn pathology throughout the brain beyond dopamine neurons in a prion-like manner. Targeting propagation of such α-syn aggregates is of high interest but requires identifying pathways involving in this process. Evidence from previous Alzheimer's disease reports suggests that EGFR may be involved in the prion-like propagation and seeding of amyloid-ß. We show here that EGFR regulates the uptake of exogenous α-syn-PFFs and the levels of endogenous α-syn in cell cultures and a mouse model of α-syn propagation, respectively. Thus, we tested the therapeutic potentials of AZD3759, a highly selective BBB-penetrating EGFR inhibitor, in a preclinical mouse model of α-syn propagation. AZD3759 decreases activated EGFR levels in the brain and reduces phosphorylated α-synuclein (pSyn) pathology in brain sections, including striatum and SN. As AZD3759 is already in the clinic, this paper's results suggest a possible repositioning of AZD3759 as a disease-modifying approach for PD.

Development of an α-synuclein knockdown peptide and evaluation of its efficacy in Parkinson's disease models.

Convincing evidence supports the premise that reducing α-synuclein levels may be an effective therapy for Parkinson's disease (PD); however, there has been lack of a clinically applicable α-synuclein reducing therapeutic strategy. This study was undertaken to develop a blood-brain barrier and plasma membrane-permeable α-synuclein knockdown peptide, Tat-ßsyn-degron, that may have therapeutic potential. The peptide effectively reduced the level of α-synuclein via proteasomal degradation both in cell cultures and in animals. Tat-ßsyn-degron decreased α-synuclein aggregates and microglial activation in an α-synuclein pre-formed fibril model of spreading synucleinopathy in transgenic mice overexpressing human A53T α-synuclein. Moreover, Tat-ßsyn-degron reduced α-synuclein levels and significantly decreased the parkinsonian toxin-induced neuronal damage and motor impairment in a mouse toxicity model of PD. These results show the promising efficacy of Tat-ßsyn-degron in two different animal models of PD and suggest its potential use as an effective PD therapeutic that directly targets the disease-causing process.

DCC Receptors Drive Prefrontal Cortex Maturation by Determining Dopamine Axon Targeting in Adolescence.

BACKGROUND: Dopaminergic input to the prefrontal cortex (PFC) increases throughout adolescence and, by establishing precisely localized synapses, calibrates cognitive function. However, why and how mesocortical dopamine axon density increases across adolescence remains unknown. METHODS: We used a developmental application of axon-initiated recombination to label and track the growth of dopamine axons across adolescence in mice. We then paired this recombination with cell-specific knockdown of the netrin-1 receptor DCC to determine its role in adolescent dopamine axon growth. We then assessed how altering adolescent PFC dopamine axon growth changes the structural and functional development of the PFC by quantifying pyramidal neuron morphology and cognitive performance. RESULTS: We show, for the first time, that dopamine axons continue to grow from the striatum to the PFC during adolescence. Importantly, we discover that DCC, a guidance cue receptor, controls the extent of this protracted growth by determining where and when dopamine axons recognize their final target. When DCC-dependent adolescent targeting events are disrupted, dopamine axons continue to grow ectopically from the nucleus accumbens to the PFC and profoundly change PFC structural and functional development. This leads to alterations in cognitive processes known to be impaired across psychiatric conditions. CONCLUSIONS: The prolonged growth of dopamine axons represents an extraordinary period for experience to influence their adolescent trajectory and predispose to or protect against psychopathology. DCC receptor signaling in dopamine neurons is a molecular link where genetic and environmental factors may interact in adolescence to influence the development and function of the prefrontal cortex.

Homeostatic regulation through GABA and acetylcholine muscarinic receptors of motor trigeminal neurons following sleep deprivation.

Muscle tone is regulated across sleep-wake states, being maximal in waking, reduced in slow wave sleep (SWS) and absent in paradoxical or REM sleep (PS or REMS). Such changes in tone have been recorded in the masseter muscles and shown to correspond to changes in activity and polarization of the trigeminal motor 5 (Mo5) neurons. The muscle hypotonia and atonia during sleep depend in part on GABA acting upon both GABAA and GABAB receptors (Rs) and acetylcholine (ACh) acting upon muscarinic 2 (AChM2) Rs. Here, we examined whether Mo5 neurons undergo homeostatic regulation through changes in these inhibitory receptors following prolonged activity with enforced waking. By immunofluorescence, we assessed that the proportion of Mo5 neurons positively stained for GABAARs was significantly higher after sleep deprivation (SD, ~65%) than sleep control (SC, ~32%) and that the luminance of the GABAAR fluorescence was significantly higher after SD than SC and sleep recovery (SR). Although, all Mo5 neurons were positively stained for GABABRs and AChM2Rs (100%) in all groups, the luminance of these receptors was significantly higher following SD as compared to SC and SR. We conclude that the density of GABAA, GABAB and AChM2 receptors increases on Mo5 neurons during SD. The increase in these receptors would be associated with increased inhibition in the presence of GABA and ACh and thus a homeostatic down-scaling in the excitability of the Mo5 neurons after prolonged waking and resulting increased susceptibility to muscle hypotonia or atonia along with sleep.

Homeostatic Changes in GABA and Acetylcholine Muscarinic Receptors on GABAergic Neurons in the Mesencephalic Reticular Formation following Sleep Deprivation.

We have examined whether GABAergic neurons in the mesencephalic reticular formation (RFMes), which are believed to inhibit the neurons in the pons that generate paradoxical sleep (PS or REMS), are submitted to homeostatic regulation under conditions of sleep deprivation (SD) by enforced waking during the day in mice. Using immunofluorescence, we investigated first, by staining for c-Fos, whether GABAergic RFMes neurons are active during SD and then, by staining for receptors, whether their activity is associated with homeostatic changes in GABAA or acetylcholine muscarinic type 2 (AChM2) receptors (Rs), which evoke inhibition. We found that a significantly greater proportion of the GABAergic neurons were positively stained for c-Fos after SD (∼27%) as compared to sleep control (SC; ∼1%) and sleep recovery (SR; ∼6%), suggesting that they were more active during waking with SD and less active or inactive during sleep with SC and SR. The density of GABAARs and AChM2Rs on the plasma membrane of the GABAergic neurons was significantly increased after SD and restored to control levels after SR. We conclude that the density of these receptors is increased on RFMes GABAergic neurons during presumed enhanced activity with SD and is restored to control levels during presumed lesser or inactivity with SR. Such increases in GABAAR and AChM2R with sleep deficits would be associated with increased susceptibility of the wake-active GABAergic neurons to inhibition from GABAergic and cholinergic sleep-active neurons and to thus permitting the onset of sleep and PS with muscle atonia.

Homeostatic Changes in GABA and Glutamate Receptors on Excitatory Cortical Neurons during Sleep Deprivation and Recovery.

Neuronal activity is regulated in a homeostatic manner through changes in inhibitory GABA and excitatory glutamate (Glu) AMPA (A) receptors (GluARs). Using immunofluorescent staining, we examined whether calcium/calmodulin-dependent protein kinase IIα (CaMKIIα)-labeled (+) excitatory neurons in the barrel cortex undergo such homeostatic regulation following enforced waking with associated cortical activation during the day when mice normally sleep the majority of the time. Sleep deprived mice were prevented from falling asleep by unilateral whisker stimulation and sleep recovery (SR) mice allowed to sleep freely following deprivation. In parallel with changes in c-Fos reflecting changes in activity, (ß2-3 subunits of) GABAA Rs were increased on the membrane of CaMKIIα+ neurons with enforced waking and returned to baseline levels with SR in barrel cortex on sides both contra- and ipsilateral to the whisker stimulation. The GABAAR increase was correlated with increased gamma electroencephalographic (EEG) activity across conditions. On the other hand, (GluA1 subunits of) AMPA Rs were progressively removed from the membrane of CaMKIIα+ neurons by (Rab5+) early endosomes during enforced waking and returned to the membrane by (Rab11+) recycling endosomes during SR. The internalization of the GluA1Rs paralleled the expression of Arc, which mediates homeostatic regulation of AMPA receptors through an endocytic pathway. The reciprocal changes in GluA1Rs relative to GABAARs suggest homeostatic down-scaling during enforced waking and sensory stimulation and restorative up-scaling during recovery sleep. Such homeostatic changes with sleep-wake states and their associated cortical activities could stabilize excitability and activity in excitatory cortical neurons.

GABA Receptors on Orexin and Melanin-Concentrating Hormone Neurons Are Differentially Homeostatically Regulated Following Sleep Deprivation.

Though overlapping in distribution through the hypothalamus, orexin (Orx) and melanin-concentrating hormone (MCH) neurons play opposite roles in the regulation of sleep-wake states. Orx neurons discharge during waking, whereas MCH neurons discharge during sleep. In the present study, we examined in mice whether GABAA and GABAB receptors (Rs) are present on Orx and MCH neurons and might undergo differential changes as a function of their different activities following sleep deprivation (SD) and sleep recovery (SR). Applying quantitative stereological image analysis to dual-immunofluorescent stained sections, we determined that the proportion of Orx neurons positively immunostained for GABAARs was significantly higher following SD (∼48%) compared with sleep control (SC; ∼24%) and SR (∼27%), and that the luminance of the GABAARs was significantly greater. In contrast, the average proportion of the MCH neurons immunostained for GABAARs was insignificantly lower following SD (∼43%) compared with SC (∼54%) and SR (56%), and the luminance of the GABAARs was significantly less. Although, GABABRs were observed in all Orx and MCH neurons (100%), the luminance of these receptors was differentially altered following SD. The intensity of GABABRs in the Orx neurons was significantly greater after SD than after SC and SR, whereas that in the MCH neurons was significantly less. The present results indicate that GABA receptors undergo dynamic and differential changes in the wake-active Orx neurons and the sleep-active MCH neurons as a function of and homeostatic adjustment to their preceding activity and sleep-wake state.