Regulator of G Protein Signaling 14 protein expression profile in the adult mouse brain.

Regulator of G protein signaling 14 (RGS14) is a multifunctional signaling protein that serves as a natural suppressor of synaptic plasticity in the mouse brain. Our previous studies showed that RGS14 is highly expressed in postsynaptic dendrites and spines of pyramidal neurons in hippocampal area CA2 of the developing mouse brain. However, our more recent work with adult rhesus macaque brain shows that RGS14 is found in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra, and amygdala in the adult rhesus monkey brain. In the mouse brain, we also have observed RGS14 protein in discrete limbic regions linked to reward behavior and addiction, including the central amygdala and the nucleus accumbens, but a comprehensive mapping of RGS14 protein expression in the adult mouse brain is lacking. Here, we report that RGS14 is more broadly expressed in mouse brain than previously known. Intense RGS14 staining is observed in specific neuron populations of the hippocampal formation, amygdala, septum, bed nucleus of stria terminalis and ventral striatum/nucleus accumbens. RGS14 is also observed in axon fiber tracts including the dorsal fornix, fimbria, stria terminalis, and the ventrohippocampal commissure. Moderate RGS14 staining is observed in various other adjacent regions not previously reported. These findings show that RGS14 is expressed in brain regions that govern aspects of core cognitive functions such as sensory perception, emotion, memory, motivation, and execution of actions, and suggests that RGS14 may serve to suppress plasticity and filter inputs in these brain regions to set the overall tone on experience-to-action processes.

Decreased cellular excitability of pyramidal tract neurons in primary motor cortex leads to paradoxically increased network activity in simulated parkinsonian motor cortex.

Decreased excitability of pyramidal tract neurons in layer 5B (PT5B) of primary motor cortex (M1) has recently been shown in a dopamine-depleted mouse model of parkinsonism. We hypothesized that decreased PT5B neuron excitability would substantially disrupt oscillatory and non-oscillatory firing patterns of neurons in layer 5 (L5) of primary motor cortex (M1). To test this hypothesis, we performed computer simulations using a previously validated computer model of mouse M1. Inclusion of the experimentally identified parkinsonism-associated decrease of PT5B excitability into our computational model produced a paradoxical increase in rest-state PT5B firing rate, as well as an increase in beta-band oscillatory power in local field potential (LFP). In the movement-state, PT5B population firing and LFP showed reduced beta and increased high-beta, low-gamma activity of 20-35 Hz in the parkinsonian, but not in control condition. The appearance of beta-band oscillations in parkinsonism would be expected to disrupt normal M1 motor output and contribute to motor activity deficits seen in patients with Parkinson's disease (PD).

Ultrastructural analysis of nigrostriatal dopaminergic terminals in a knockin mouse model of DYT1 dystonia.

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia.

Tyrosinase-induced neuromelanin accumulation triggers rapid dysregulation and degeneration of the mouse locus coeruleus.

Motor symptoms in Parkinson's disease (PD) are caused by degeneration of dopamine (DA) neurons of the substantia nigra (SN), while early non-motor symptoms such as anxiety and sleep disturbances are likely mediated by dysfunction of locus coeruleus (LC) norepinephrine (NE) neurons. The LC develops α-synuclein pathology prior to SN DA neurons in PD, and later undergoes degeneration, but the mechanisms responsible for its vulnerability are unknown. The SN and LC are the only structures in the brain that produces appreciable amounts of neuromelanin (NM), a dark cytoplasmic pigment. It has been proposed that NM initially plays a protective role by sequestering toxic catecholamine metabolites and heavy metals, but may become harmful during aging and PD as they overwhelm cellular machinery and are released during neurodegeneration. Rodents do not naturally produce NM, limiting the study of causal relationships between NM and PD-associated LC pathology. Adapting a viral-mediated approach for expression of human tyrosinase, the enzyme responsible for peripheral melanin production, we successfully promoted pigmentation in mouse LC neurons that recapitulates key features of endogenous NM found in primates, including eumelanin and pheomelanin, lipid droplets, and a double-membrane encasement. Pigment expression results in mild neurodegeneration, reduced NE levels, transcriptional changes, and novelty-induced anxiety phenotypes as early as 1-week post-injection. By 6-weeks, NM accumulation is associated with severe LC neurodegeneration and a robust neuroinflammatory response. These phenotypes are reminiscent of LC dysfunction in PD, validating this model for studying the consequences of pigment accumulation in the LC as it relates to neurodegenerative disease.

RGS14 is neuroprotective against seizure-induced mitochondrial oxidative stress and pathology in hippocampus.

RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. There, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies show that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Utilizing proteomics, we saw loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro . As a readout of oxidative stress, we found RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we observed worse neuronal injury in area CA3 (but none in CA2 or CA1), and a lack of microgliosis in CA1 and CA2 compared to WT. Together, our data demonstrates a newly appreciated neuroprotective role for RGS14 against intense seizure activity in hippocampus. Our findings are consistent with a model where, after seizure, RGS14 is upregulated to support mitochondrial function and prevent oxidative stress in CA2 PCs, limit seizure onset and hippocampal neuronal injury, and promote microglial activation in hippocampus.

Levodopa responsive freezing of gait is associated with reduced norepinephrine transporter binding in Parkinson's disease.

BACKGROUND: Freezing of gait (FOG) is a major cause of falling in Parkinson's disease (PD) and can be responsive or unresponsive to levodopa. Pathophysiology is poorly understood. OBJECTIVE: To examine the link between noradrenergic systems, the development of FOG in PD and its responsiveness to levodopa. METHODS: We examined norepinephrine transporter (NET) binding via brain positron emission tomography (PET) to evaluate changes in NET density associated with FOG using the high affinity selective NET antagonist radioligand [11C]MeNER (2S,3S)(2-[α-(2-methoxyphenoxy)benzyl]morpholine) in 52 parkinsonian patients. We used a rigorous levodopa challenge paradigm to characterize PD patients as non-freezing (NO-FOG, N = 16), levodopa responsive freezing (OFF-FOG, N = 10), and levodopa-unresponsive freezing (ONOFF-FOG, N = 21), and also included a non-PD FOG group, primary progressive freezing of gait (PP-FOG, N = 5). RESULTS: Linear mixed models identified significant reductions in whole brain NET binding in the OFF-FOG group compared to the NO-FOG group (-16.8%, P = 0.021) and regionally in the frontal lobe, left and right thalamus, temporal lobe, and locus coeruleus, with the strongest effect in right thalamus (P = 0.038). Additional regions examined in a post hoc secondary analysis including the left and right amygdalae confirmed the contrast between OFF-FOG and NO-FOG (P = 0.003). A linear regression analysis identified an association between reduced NET binding in the right thalamus and more severe New FOG Questionnaire (N-FOG-Q) score only in the OFF-FOG group (P = 0.022). CONCLUSION: This is the first study to examine brain noradrenergic innervation using NET-PET in PD patients with and without FOG. Based on the normal regional distribution of noradrenergic innervation and pathological studies in the thalamus of PD patients, the implications of our findings suggest that noradrenergic limbic pathways may play a key role in OFF-FOG in PD. This finding could have implications for clinical subtyping of FOG as well as development of therapies.

Characterization of the GfaABC1D promoter to selectively target astrocytes in the rhesus macaque brain.

BACKGROUND: The study of astrocytic functions in non-human primates (NHPs) has been hampered by the lack of genetic tools to selectively target astrocytes. Viral vectors with selective and efficient transduction of astrocytes could be a potent tool to express marker proteins, modulators, or sensors in NHP astrocytes, but the availability of thoroughly characterized astrocytic selective promoter sequences to use in these species remains extremely limited. NEW METHOD: We describe the specificity and efficiency of an astrocyte-specific promoter, GfaABC1D in the brain of the rhesus macaque, with emphasis in basal ganglia regions. AAV5-pZac2.1-GfaABC1D-tdTomato was locally injected into the globus pallidus external segment (GPe) and putamen. The extent, efficiency, and specificity of transduction was analyzed with immunohistochemistry at the light and electron microscope levels. RESULTS: The GfaABC1D promoter directed the expression of tdTomato in an astrocyte-specific manner in directly or indirectly targeted regions (including both segments of the globus pallidus, putamen, subthalamic nucleus and cortex). COMPARISON WITH EXISTING METHODS: Due to its small size, the GfaABC1D promoter is advantageous over other previously used glial fibrillary acidic protein-based promoter sequences, facilitating its use to drive expression of various transgenes in adeno-associated viruses (AAV) or other viral vectors. CONCLUSION: GfaABC1D is an efficient promoter that selectively targets astrocytes in the monkey basal ganglia and expands the viral vector toolbox to study astrocytic functions in non-human primates.

Cortical Serotonergic and Catecholaminergic Denervation in MPTP-Treated Parkinsonian Monkeys.

Decreased cortical serotonergic and catecholaminergic innervation of the frontal cortex has been reported at early stages of Parkinson's disease (PD). However, the limited availability of animal models that exhibit these pathological features has hampered our understanding of the functional significance of these changes during the course of the disease. In the present study, we assessed longitudinal changes in cortical serotonin and catecholamine innervation in motor-symptomatic and asymptomatic monkeys chronically treated with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Densitometry and unbiased stereological techniques were used to quantify changes in serotonin and tyrosine hydroxylase (TH) immunoreactivity in frontal cortices of 3 control monkeys and 3 groups of MPTP-treated monkeys (motor-asymptomatic [N = 2], mild parkinsonian [N = 3], and moderate parkinsonian [N = 3]). Our findings revealed a significant decrease (P < 0.001) in serotonin innervation of motor (Areas 4 and 6), dorsolateral prefrontal (Areas 9 and 46), and limbic (Areas 24 and 25) cortical areas in motor-asymptomatic MPTP-treated monkeys. Both groups of symptomatic MPTP-treated animals displayed further serotonin denervation in these cortical regions (P < 0.0001). A significant loss of serotonin-positive dorsal raphe neurons was found in the moderate parkinsonian group. On the other hand, the intensity of cortical TH immunostaining was not significantly affected in motor asymptomatic MPTP-treated monkeys, but underwent a significant reduction in the moderate symptomatic group (P < 0.05). Our results indicate that chronic intoxication with MPTP induces early pathology in the corticopetal serotonergic system, which may contribute to early non-motor symptoms in PD.

Cerebral Aß deposition in an Aß-precursor protein-transgenic rhesus monkey.

With the ultimate goal of developing a more representative animal model of Alzheimer's disease (AD), two female amyloid-ß-(Aß) precursor protein-transgenic (APPtg) rhesus monkeys were generated by lentiviral transduction of the APP gene into rhesus oocytes, followed by in vitro fertilization and embryo transfer. The APP-transgene included the AD-associated Swedish K670N/M671L and Indiana V717F mutations (APPSWE/IND) regulated by the human polyubiquitin-C promoter. Overexpression of APP was confirmed in lymphocytes and brain tissue. Upon sacrifice at 10 years of age, one of the monkeys had developed Aß plaques and cerebral Aß-amyloid angiopathy in the occipital, parietal, and caudal temporal neocortices. The induction of Aß deposition more than a decade prior to its usual emergence in the rhesus monkey supports the feasibility of creating a transgenic nonhuman primate model for mechanistic analyses and preclinical testing of treatments for Alzheimer's disease and cerebrovascular amyloidosis.

Glutamate Delta-1 Receptor Regulates Inhibitory Neurotransmission in the Nucleus Accumbens Core and Anxiety-Like Behaviors.

Glutamate delta-1 receptor (GluD1) is a member of the ionotropic glutamate receptor family expressed at excitatory synapses and functions as a synaptogenic protein by interacting with presynaptic neurexin. We have previously shown that GluD1 plays a role in the maintenance of excitatory synapses in a region-specific manner. Loss of GluD1 leads to reduced excitatory neurotransmission in medium spiny neurons (MSNs) in the dorsal striatum, but not in the ventral striatum (both core and shell of the nucleus accumbens (NAc)). Here, we found that GluD1 loss leads to reduced inhibitory neurotransmission in MSNs of the NAc core as evidenced by a reduction in the miniature inhibitory postsynaptic current frequency and amplitude. Presynaptic effect of GluD1 loss was further supported by an increase in paired pulse ratio of evoked inhibitory responses indicating reduced release probability. Furthermore, analysis of GAD67 puncta indicated a reduction in the number of putative inhibitory terminals. The changes in mIPSC were independent of cannabinoid or dopamine signaling. A role of feed-forward inhibition was tested by selective ablation of GluD1 from PV neurons which produced modest reduction in mIPSCs. Behaviorally, local ablation of GluD1 from NAc led to hypolocomotion and affected anxiety- and depression-like behaviors. When GluD1 was ablated from the dorsal striatum, several behavioral phenotypes were altered in opposite manner compared to GluD1 ablation from NAc. Our findings demonstrate that GluD1 regulates inhibitory neurotransmission in the NAc by a combination of pre- and postsynaptic mechanisms which is critical for motor control and behaviors relevant to neuropsychiatric disorders.

Intracerebroventricular Administration of AAV9-PHP.B SYN1-EmGFP Induces Widespread Transgene Expression in the Mouse and Monkey Central Nervous System.

Viral vectors made from adeno-associated virus (AAV) have emerged as preferred tools in basic and translational neuroscience research to introduce or modify genetic material in cells of interest. The use of viral vectors is particularly attractive in nontransgenic species, such as nonhuman primates. Injection of AAV solutions into the cerebrospinal fluid is an effective method to achieve a broad distribution of a transgene in the central nervous system. In this study, we conducted injections of AAV9-PHP.B, a recently described AAV capsid mutant, in the lateral ventricle of mice and rhesus macaques. To enhance the expression of the transgene (the tag protein emerald green fluorescent protein [EmGFP]), we used a gene promoter that confers high neuron-specific expression of the transgene, the human synapsin 1 (SYN1) promoter. The efficacy of the viral vector was first tested in mice. Our results show that intracerebroventricular injections of AAV9-PHP.B SYN1-EmGFP-woodchuck hepatitis virus posttranscriptional regulatory element resulted in neuronal EmGFP expression throughout the mice and monkey brains. We have provided a thorough characterization of the brain regions expressing EmGFP in both species. EmGFP was observed in neuronal cell bodies over the whole cerebral cortex and in the cerebellum, as well as in some subcortical regions, including the striatum and hippocampus. We also observed densely labeled neuropil in areas known to receive projections from these regions. Double fluorescence studies demonstrated that EmGFP was expressed by several types of neurons throughout the mouse and monkey brain. Our results demonstrate that a single injection in the lateral ventricle is an efficient method to obtain transgene expression in many cortical and subcortical regions, obviating the need of multiple intraparenchymal injections to cover large brain areas. The use of intraventricular injections of AAV9-PHP.B SYN1-EmGFP could provide a powerful approach to transduce widespread areas of the brain and may contribute to further development of methods to genetically target-specific populations of neurons.

Common and distinct neural trends of allocentric and egocentric spatial coding: An ALE meta-analysis.

The uniqueness of neural processes between allocentric and egocentric spatial coding has been controversial. The distinctive paradigms used in previous studies for manipulating spatial coding could have attributed for the inconsistent results. This study was aimed to generate converging evidence from previous functional brain imaging experiments for collating neural substrates associated with these two types of spatial coding. An additional aim was to test whether test-taking processes would have influenced the results. We obtained coordinate-based functional neuroimaging data for 447 subjects and performed activation likelihood estimation (ALE) meta-analysis. Among the 28 experiments, the results indicate two common clusters of convergence. They were the right precuneus and the right superior frontal gyrus as parts of the parieto-frontal circuit. Between-type differences were in the parieto-occipital circuit, with allocentric showing convergence in the superior occipital gyrus (SOG) cluster compared with egocentric showing convergence in the middle occipital gyrus (MOG) cluster. Task-specific influences were only found in allocentric spatial coding. Spatial judgment-oriented tasks seem to increase the demands on manipulating spatial relationships among the visual objects, while spatial navigation tasks seem to increase the demands on maintaining object representations. Our findings address the theoretical controversies on spatial coding that both the allocentric and egocentric types are common in their processes mediated by the parieto-frontal network, while unique and additional processes in the allocentric type are mediated by the parieto-occipital network. The positive results on possible task-specific confound offer insights into the future design of spatial tasks for eliciting spatial coding processes.

Comparative Ultrastructural Analysis of Thalamocortical Innervation of the Primary Motor Cortex and Supplementary Motor Area in Control and MPTP-Treated Parkinsonian Monkeys.

The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.

Glutamatergic inputs to GABAergic interneurons in the motor thalamus of control and parkinsonian monkeys.

The primate ventral motor thalamus contains a large number of GABAergic interneurons of poorly understood function and anatomical connectivity. Glutamatergic inputs to these cells arise predominantly from corticothalamic (in both basal ganglia- and cerebellar-receiving ventral motor thalamic territories; BGMT and CBMT, respectively) and cerebellothalamic terminals (in CBMT). In Parkinson's disease patients and animal models, neuronal activity is abnormal within both BGMT and CBMT. Historically, such motor thalamic dysregulation has been largely attributed to changes in inhibitory tone from the basal ganglia output nuclei, ignoring the potential role of other thalamic inputs in such processes, particularly within the CBMT, which is largely devoid of direct basal ganglia afferents. We have recently reported changes in the abundance and structural morphology of corticothalamic terminals in BGMT of parkinsonian monkeys. In this study, we assessed potential changes in the prevalence of cortical (vesicular glutamate transporter 1-positive, vGluT1-positive) and subcortical (vGluT2-positive) glutamatergic inputs in contact with GABAergic interneurons in BGMT and CBMT of MPTP-treated parkinsonian monkeys. Our findings revealed that interneurons represent a major target of both sets of glutamatergic terminals. In both BGMT and CBMT of control and parkinsonian monkeys, 29%-38% of total asymmetric axodendritic synapses (putative glutamatergic) were formed by vGluT1-positive terminals and 11%-17% of total vGluT1-positive terminals targeted dendrites of GABAergic interneurons. In CBMT, 16%-18% of asymmetric synaptic inputs on interneurons involved vGluT2-containing terminals. No major differences in the extent of glutamatergic innervation of thalamic GABAergic interneurons were found between control and parkinsonian monkeys.

Ultrastructural localization of glutamate delta 1 (GluD1) receptor immunoreactivity in the mouse and monkey striatum.

The glutamate receptor delta 1 (GluD1) is strongly expressed in the striatum. Knockout of GluD1 expression in striatal neurons elicits cognitive deficits and disrupts the thalamostriatal system in mice. To understand the potential role of GluD1 in the primate striatum, we compared the cellular and subcellular localization of striatal GluD1 immunoreactivity (GluD1-IR) in mice and monkeys. In both species, striatal GluD1-IR displayed a patchy pattern of distribution in register with the striosome/matrix compartmentation, but in an opposite fashion. While GluD1 was more heavily expressed in the striosomes than the matrix in the monkey caudate nucleus, the opposite was found in the mouse striatum. At the electron microscopic level, GluD1-IR was preferentially expressed in dendritic shafts (47.9 ± 1.2%), followed by glia (37.7 ± 2.5%), and dendritic spines (14.3 ± 2.6%) in the matrix of the mouse striatum. This pattern was not statistically different from the labeling in the striosome and matrix compartments of the monkey caudate nucleus, with the exception of a small amount of GluD1-positive unmyelinated axons and axon terminals in the primate striatum. Immunogold staining revealed synaptic and perisynaptic GluD1 labeling at putative axo-dendritic and axo-spinous glutamatergic synapses, and intracellular labeling on the surface of mitochondria. Confocal microscopy showed that GluD1 is preferentially colocalized with thalamic over cortical terminals in both the striosome and matrix compartments. These data provide the anatomical substrate for a deeper understanding of GluD1 regulation of striatal glutamatergic synapses, but also suggest possible extrasynaptic, glial, and mitochondrial GluD1 functions.