Empagliflozin attenuates arrhythmogenesis in diabetic cardiomyopathy by normalizing intracellular Ca2+ handling in ventricular cardiomyocytes.

Diabetic cardiomyopathy has been reported to increase the risk of fatal ventricular arrhythmia. The beneficial effects of the selective sodium-glucose cotransporter-2 inhibitor have not been fully examined in the context of antiarrhythmic therapy, especially its direct cardioprotective effects despite the negligible SGLT2 expression in cardiomyocytes. We aimed to examine the antiarrhythmic effects of empagliflozin (EMPA) treatment on diabetic cardiomyocytes, with a special focus on Ca2+ handling. We conducted echocardiography and hemodynamic studies and studied electrophysiology, Ca2+ handling, and protein expression in C57BLKS/J-leprdb/db mice (db/db mice) and their nondiabetic lean heterozygous Leprdb/+ littermates (db/+ mice). Preserved systolic function with diastolic dysfunction was observed in 16-wk-old db/db mice. During arrhythmia induction, db/db mice had significantly increased premature ventricular complexes (PVCs) than controls, which was attenuated by EMPA. In protein expression analyses, calmodulin-dependent protein kinase II (CaMKII) Thr287 autophosphorylation and CaMKII-dependent RyR2 phosphorylation (S2814) were significantly increased in diabetic hearts, which were inhibited by EMPA. In addition, global O-GlcNAcylation significantly decreased with EMPA treatment. Furthermore, EMPA significantly inhibited ventricular cardiomyocyte glucose uptake. Diabetic cardiomyocytes exhibited increased spontaneous Ca2+ events and decreased sarcoplasmic reticulum (SR) Ca2+ content, along with impaired Ca2+ transient, all of which normalized with EMPA treatment. Notably, most EMPA-induced improvements in Ca2+ handling were abolished by the addition of an O-GlcNAcase (OGA) inhibitor. In conclusion, EMPA attenuated ventricular arrhythmia inducibility by normalizing the intracellular Ca2+ handling, and we speculated that this effect was, at least partly, due to the inhibition of O-GlcNAcylation via the suppression of glucose uptake into cardiomyocytes.NEW & NOTEWORTHY SGLT2is are known to improve cardiovascular outcomes regardless of the presence of diabetes and decrease traditional cardiovascular risk factors. We demonstrated, for the first time, that EMPA inhibited PVCs by normalizing Ca2+ handling in diabetic mice. Our data suggest that the effects of SGLT2is on calcium handling may occur because of suppression of O-GlcNAcylation through inhibition of glucose uptake and not because of NHE inhibition, as previously suggested.

Acetylcholine from the nucleus basalis magnocellularis facilitates the retrieval of well-established memory.

Retrieval deficit of long-term memory is a cardinal symptom of dementia and has been proposed to associate with abnormalities in the central cholinergic system. Difficulty in the retrieval of memory is experienced by healthy individuals and not limited to patients with neurological disorders that result in forgetfulness. The difficulty of retrieving memories is associated with various factors, such as how often the event was experienced or remembered, but it is unclear how the cholinergic system plays a role in the retrieval of memory formed by a daily routine (accumulated experience). To investigate this point, we trained rats moderately (for a week) or extensively (for a month) to detect a visual cue in a two-alternative forced-choice task. First, we confirmed the well-established memory in the extensively trained group was more resistant to the retrieval problem than recently acquired memory in the moderately trained group. Next, we tested the effect of a cholinesterase inhibitor, donepezil, on the retrieval of memory after a long no-task period in extensively trained rats. Pre-administration of donepezil improved performance and reduced the latency of task initiation compared to the saline-treated group. Finally, we lesioned cholinergic neurons of the nucleus basalis magnocellularis (NBM), which project to the entire neocortex, by injecting the cholinergic toxin 192 IgG-saporin. NBM-lesioned rats showed severely impaired task initiation and performance. These abilities recovered as the trials progressed, though they never reached the level observed in rats with intact NBM. These results suggest that acetylcholine released from the NBM contributes to the retrieval of well-established memory developed by a daily routine.

Perineuronal Nets in the Deep Cerebellar Nuclei Regulate GABAergic Transmission and Delay Eyeblink Conditioning.

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.

Parvalbumin-producing striatal interneurons receive excitatory inputs onto proximal dendrites from the motor thalamus in male mice.

In rodents, the dorsolateral striatum regulates voluntary movement by integrating excitatory inputs from the motor-related cerebral cortex and thalamus to produce contingent inhibitory output to other basal ganglia nuclei. Striatal parvalbumin (PV)-producing interneurons receiving this excitatory input then inhibit medium spiny neurons (MSNs) and modify their outputs. To understand basal ganglia function in motor control, it is important to reveal the precise synaptic organization of motor-related cortical and thalamic inputs to striatal PV interneurons. To examine which domains of the PV neurons receive these excitatory inputs, we used male bacterial artificial chromosome transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein in PV neurons. An anterograde tracing study with the adeno-associated virus vector combined with immunodetection of pre- and postsynaptic markers visualized the distribution of the excitatory appositions on PV dendrites. Statistical analysis revealed that the density of thalamostriatal appositions along the dendrites was significantly higher on the proximal than distal dendrites. In contrast, there was no positional preference in the density of appositions from axons of the dorsofrontal cortex. Population observations of thalamostriatal and corticostriatal appositions by immunohistochemistry for pathway-specific vesicular glutamate transporters confirmed that thalamic inputs preferentially, and cortical ones less preferentially, made apposition on proximal dendrites of PV neurons. This axodendritic organization suggests that PV neurons produce fast and reliable inhibition of MSNs in response to thalamic inputs and process excitatory inputs from motor cortices locally and plastically, possibly together with other GABAergic and dopaminergic dendritic inputs, to modulate MSN inhibition.

Quantitative Analyses of the Projection of Individual Neurons from the Midline Thalamic Nuclei to the Striosome and Matrix Compartments of the Rat Striatum.

A fundamental organizing principle of the striatum is the striosome/matrix system that is defined by inputs/outputs and neurochemical markers. The thalamostriatal projection is highly heterogeneous originating in many subnuclei of the thalamus including the midline (ML) and intralaminar (IL) nuclei. We examined the dendritic morphology and axonal trajectory of 15 ML and 11 IL neurons by single-neuron labeling with viral vectors in combination with mu-opioid receptor immunostaining in rat brains. Dendritic and axonal morphology defined ML neurons as type II cells consisting of at least two subclasses according to the presence or absence of striatal axon collaterals. In the striatum, ML neurons preferentially innervated striosomes, whereas parafascicular neurons preferentially innervated the matrix. Almost all single thalamostriatal neurons favoring striosome or matrix compartments also innervated the cerebral cortical areas that supplied cortical input to the same striatal compartment. We thus revealed that thalamostriatal projections are highly organized 1) by the similarity in morphological characteristics and 2) their preference for the striatal compartments and cortical areas. These findings demonstrate that the functional properties of striatal compartments are influenced by both their cortical and thalamic afferents presumably with a different time latency and support selective dynamics for the striosome and matrix compartments.

Results of Questionnaire Survey on Gross Anatomy Education (March 2014).

To understand the current situation of gross anatomy education anatomy classes. Regarding the influence of increased enrollment and to promote sharing of information on its improvement, we capacity in medical schools, many respondents were worried about conducted a questionnaire survey on gross anatomy education the impact on research activities due to the increase in teaching in September 2013. In most medical and dental schools, gross workload without expanding in teaching staff. In some schools, anatomy courses were offered to second-year students. The owing to the limitations of the facilities or the number of donated average numbers of gross anatomy practices were 34.6 in medical bodies, the number of students per cadaver had to be increased. schools and 27.4 in dental schools. The average total hours of We received various effective and practical measures for the practice in the curriculum was 125 in medical schools, and 97 improvement of gross anatomy education, such as improvement in dental schools. However, in about 80% of total schools, the of teaching materials and dissection methods, introduction of length of the actual gross anatomy practice was considerably lectures on clinical anatomy by clinicians, and implementation longer, because the students could not finish the work within of the second-round gross anatomy practice in the upper grades. the allotted class time. As to the effect of curriculum reform in Many respondents emphasized both the need for a training system respond to the introduction of the accreditation of medical and for young teaching staff, and the importance of opportunities for dental education programs, many respondents answered that sharing information on education. they had a minimal effect except earlier commencement of gross.

Cell type-specific inhibitory inputs to dendritic and somatic compartments of parvalbumin-expressing neocortical interneuron.

Parvalbumin (PV)-producing fast-spiking neurons are well known to generate gamma oscillation by mutual chemical and electrical connections in the neocortex. Although it was clearly demonstrated that PV neurons form a dense gap junction network with each other not only at the proximal sites but also at the distal dendrites, comprehensive quantitative data on the chemical connections are still lacking. To elucidate the connectivity, we investigated inhibitory inputs to PV neurons in the somatosensory cortex, using the transgenic mice in which the dendrites and cell bodies of PV neurons were clearly visualized. We first examined GABAergic inputs to PV neurons by labeling postsynaptic and presynaptic sites with the immunoreactivities for gephyrin and vesicular GABA transporter. The density of GABAergic inputs was highest on the cell bodies, and almost linearly decreased to the distal dendrites. We then investigated inhibitory inputs from three distinct subgroups of GABAergic interneurons by visualizing the axon terminals immunopositive for PV, somatostatin (SOM), or vasoactive intestinal polypeptide (VIP). PV and SOM inputs were frequently located on the dendrites with the ratio of 2.5:1, but much less on the cell bodies. By contrast, VIP inputs clearly preferred the cell bodies to the dendrites. Consequently, the dendritic and somatic compartments of PV neurons received ∼60 and 62% of inhibitory inputs from PV and VIP neurons, respectively. This compartmental organization of inhibitory inputs suggests that PV neurons, together with gap junctions, constitute mutual connections at the dendrites, and that their activities are negatively controlled by the somatic inputs of VIP neurons.

Pre- and Postembedding Immunoelectron Microscopy to Analyze the Central Nervous System.

Immunocytochemistry, a method of delineating the subcellular localization of target proteins, was developed from immunohistochemistry. In principle, proteins are labeled using an antigen-antibody reaction. In order to observe under an electron microscope, the reaction product must scatter the electron beam with sufficient contrast while it is necessary to have an amplifying label that can withstand the observation. We have some detailed tips on making electron microscope samples to achieve this objective, and we would be happy to help you.

Volume electron microscopy for genetically and molecularly defined neural circuits.

The brain networks responsible for adaptive behavioral changes are based on the physical connections between neurons. Light and electron microscopy have long been used to study neural projections and the physical connections between neurons. Volume electron microscopy has recently expanded its scale of analysis due to methodological advances, resulting in complete wiring maps of neurites in a large volume of brain tissues and even entire nervous systems in a growing number of species. However, structural approaches frequently suffer from inherent limitations in which elements in images are identified solely by morphological criteria. Recently, an increasing number of tools and technologies have been developed to characterize cells and cellular components in the context of molecules and gene expression. These advancements include newly developed probes for visualization in electron microscopic images as well as correlative integration methods for the same elements across multiple microscopic modalities. Such approaches advance our understanding of interactions between specific neurons and circuits and may help to elucidate novel aspects of the basal ganglia network involving dopamine neurons. These advancements are expected to reveal mechanisms for processing adaptive changes in specific neural circuits that modulate brain functions.

Globus pallidus is not independent from striatal direct pathway neurons: an up-to-date review.

Striatal projection neurons, which are classified into two groups-direct and indirect pathway neurons, play a pivotal role in our understanding of the brain's functionality. Conventional models propose that these two pathways operate independently and have contrasting functions, akin to an "accelerator" and "brake" in a vehicle. This analogy further elucidates how the depletion of dopamine neurons in Parkinson's disease can result in bradykinesia. However, the question arises: are these direct and indirect pathways truly autonomous? Despite being distinct types of neurons, their interdependence cannot be overlooked. Single-neuron tracing studies employing membrane-targeting signals have shown that the majority of direct pathway neurons terminate not only in the output nuclei, but also in the external segment of the globus pallidus (GP in rodents), a relay nucleus of the indirect pathway. Recent studies have unveiled the existence of arkypallidal neurons, which project solely to the striatum, in addition to prototypic neurons. This raises the question of which type of GP neurons receive these striatal axon collaterals. Our morphological and electrophysiological experiments showed that the striatal direct pathway neurons may affect prototypic neurons via the action of substance P on neurokinin-1 receptors. Conversely, another research group has reported that direct pathway neurons inhibit arkypallidal neurons via GABA. Regardless of the neurotransmitter involved, it can be concluded that the GP is not entirely independent of direct pathway neurons. This review article underscores the intricate interplay between different neuronal pathways and challenges the traditional understanding of their independence.

Anterograde trans-neuronal labeling of striatal interneurons in relation to dopamine neurons in the substantia nigra pars compacta.

Recent advances in neural tracing have unveiled numerous neural circuits characterized by brain region and cell type specificity, illuminating the underpinnings of specific functions and behaviors. Dopaminergic (DA) neurons in the midbrain are highly heterogeneous in terms of gene and protein expression and axonal projections. Different cell types within the substantia nigra pars compacta (SNc) tend to project to the striatum in a cell-type-dependent manner characterized by specific topography. Given the wide and dense distribution of DA axons, coupled with a combination of synaptic and volume transmission, it remains unclear how DA release is spatially and temporally regulated, to appropriately achieve specific behaviors and functions. Our hypothesis posits that hidden rules governing synapse formation between pre-synaptic DA neuron types and striatal neuron types may modulate the effect of DA at a single-cell level. To address this conjecture, we employed adeno-associated virus serotype 1 (AAV1) to visualize the neural circuitry of DA neurons. AAV1 has emerged as a potent anatomical instrument capable of labeling and visualizing pre- and post-synaptic neurons simultaneously through anterograde trans-synaptic labeling. First, AAV1-Cre was injected into the SNc, resulting in Cre expression in both medium spiny neurons and interneurons in the striatum. Due to the potential occurrence of the retrograde transfer of AAV1, only striatal interneurons were considered for trans-synaptic or trans-neuronal labeling. Interneuron types expressing parvalbumin, choline acetyltransferase, somatostatin, or nitrogen oxide synthase exhibited Cre expression. Using a combination of AAV1-Cre and Cre-driven fluorophore expressing AAVs, striatal interneurons and the axons originating from the SNc were visualized in distinct colors. Using immunofluorescence against neurotransmitter transporters, almost all axons in the striatum visualized using this approach were confirmed to be dopaminergic. Moreover, individual DA axons established multiple appositions on the somata and proximal dendrites of interneurons. This finding suggests that irrespective of the extensive and widespread axonal arborization of DA neurons, a particular DA neuron may exert a significant influence on specific interneurons. Thus, AAV1-based labeling of the DA system can be a valuable tool to uncover the concealed rules governing these intricate relationships.

A morphological analysis of thalamocortical axon fibers of rat posterior thalamic nuclei: a single neuron tracing study with viral vectors.

The rostral sector of the posterior thalamic nuclei (POm) is, together with the ventral posterior nuclei (VP), involved in somatosensory information processing in rodents. The POm receives inputs from the spinal cord and trigeminal nuclei and projects to the primary somatosensory (S1) cortex and other cortical areas. Although thalamocortical axons of single VP neurons are well known to innervate layer (L) 4 of the S1 cortex with distinct columnar organization, those of POm neurons have not been elucidated yet. In the present study, we investigated complete axonal and dendritic arborizations of single POm neurons in rats by visualizing the processes with Sindbis viruses expressing membrane-targeted fluorescent protein. When we divided the POm into anterior and posterior parts according to calbindin immunoreactivity, dendrites of posterior POm neurons were wider but less numerous than those of anterior neurons. More interestingly, axon fibers of anterior POm neurons were preferentially distributed in L5 of the S1 cortex, whereas those of posterior neurons were principally spread in L1 with wider and sparser arborization than those of anterior neurons. These results suggest that the POm is functionally segregated into anterior and posterior parts and that the 2 parts may play different roles in somatosensory information processing.

Optogenetic activation of the ventral tegmental area-hippocampal pathway facilitates rapid adaptation to changes in spatial goals.

Animal adaptation to environmental goals to pursue rewards is modulated by dopamine. However, the role of dopamine in the hippocampus, involved in spatial navigation, remains unclear. Here, we studied dopaminergic inputs from the ventral tegmental area (VTA) to the hippocampus, focusing on spatial goal persistence and adaptation. Mice with VTA dopaminergic lesions struggled to locate and update learned reward locations in a circular maze with dynamic reward locations, emphasizing the importance of VTA dopaminergic neurons in the persistence and adaptation of spatial memory. Further, these deficits were accompanied by motor impairments or motivational loss even when dopamine receptors in the dorsal hippocampus were selectively blocked. Stimulation of VTA dopaminergic axons within the dorsal hippocampus enhanced the mice's ability to adapt to changing reward locations. These findings provide insights into the contribution of dopaminergic inputs within the hippocampus to spatial goal adaptation.

Local connections of excitatory neurons to corticothalamic neurons in the rat barrel cortex.

Corticothalamic projection neurons in the cerebral cortex constitute an important component of the thalamocortical reciprocal circuit, an essential input/output organization for cortical information processing. However, the spatial organization of local excitatory connections to corticothalamic neurons is only partially understood. In the present study, we first developed an adenovirus vector expressing somatodendritic membrane-targeted green fluorescent protein. After injection of the adenovirus vector into the ventrobasal thalamic complex, a band of layer (L) 6 corticothalamic neurons in the rat barrel cortex were retrogradely labeled. In addition to their cell bodies, fine dendritic spines of corticothalamic neurons were well visualized without the labeling of their axon collaterals or thalamocortical axons. In cortical slices containing retrogradely labeled L6 corticothalamic neurons, we intracellularly stained single pyramidal/spiny neurons of L2-6. We examined the spatial distribution of contact sites between the local axon collaterals of each pyramidal neuron and the dendrites of corticothalamic neurons. We found that corticothalamic neurons received strong and focused connections from L4 neurons just above them, and that the most numerous nearby and distant sources of local excitatory connections to corticothalamic neurons were corticothalamic neurons themselves and L6 putative corticocortical neurons, respectively. These results suggest that L4 neurons may serve as an important source of local excitatory inputs in shaping the cortical modulation of thalamic activity.

Parvalbumin-producing cortical interneurons receive inhibitory inputs on proximal portions and cortical excitatory inputs on distal dendrites.

To examine inputs to parvalbumin (PV)-producing interneurons, we generated transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein specifically in the interneurons, and completely visualized their dendrites and somata. Using immunolabeling for vesicular glutamate transporter (VGluT)1, VGluT2, and vesicular GABA transporter, we found that VGluT1-positive terminals made contacts 4- and 3.1-fold more frequently with PV-producing interneurons than VGluT2-positive and GABAergic terminals, respectively, in the primary somatosensory cortex. Even in layer 4, where VGluT2-positive terminals were most densely distributed, VGluT1-positive inputs to PV-producing interneurons were 2.4-fold more frequent than VGluT2-positive inputs. Furthermore, although GABAergic inputs to PV-producing interneurons were as numerous as VGluT2-positive inputs in most cortical layers, GABAergic inputs clearly preferred the proximal dendrites and somata of the interneurons, indicating that the sites of GABAergic inputs were more optimized than those of VGluT2-positive inputs. Simulation analysis with a PV-producing interneuron model compatible with the present morphological data revealed a plausible reason for this observation, by showing that GABAergic and glutamatergic postsynaptic potentials evoked by inputs to distal dendrites were attenuated to 60 and 87%, respectively, of those evoked by somatic inputs. As VGluT1-positive and VGluT2-positive axon terminals were presumed to be cortical and thalamic glutamatergic inputs, respectively, cortical excitatory inputs to PV-producing interneurons outnumbered the thalamic excitatory and intrinsic inhibitory inputs more than two-fold in any cortical layer. Although thalamic inputs are known to evoke about two-fold larger unitary excitatory postsynaptic potentials than cortical ones, the present results suggest that cortical inputs control PV-producing interneurons at least as strongly as thalamic inputs.

Conservation of the Direct and Indirect Pathway Dichotomy in Mouse Caudal Striatum With Uneven Distribution of Dopamine Receptor D1- and D2-Expressing Neurons.

The striatum is one of the key nuclei for adequate control of voluntary behaviors and reinforcement learning. Two striatal projection neuron types, expressing either dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R) constitute two independent output routes: the direct or indirect pathways, respectively. These pathways co-work in balance to achieve coordinated behavior. Two projection neuron types are equivalently intermingled in most striatal space. However, recent studies revealed two atypical zones in the caudal striatum: the zone in which D1R-neurons are the minor population (D1R-poor zone) and that in which D2R-neurons are the minority (D2R-poor zone). It remains obscure as to whether these imbalanced zones have similar properties on axonal projections and electrophysiology compared to other striatal regions. Based on morphological experiments in mice using immunofluorescence, in situ hybridization, and neural tracing, here, we revealed that the poor zones densely projected to the globus pallidus and substantia nigra pars lateralis, with a few collaterals in substantia nigra pars reticulata and compacta. Similar to that in other striatal regions, D1R-neurons were the direct pathway neurons. We also showed that the membrane properties of projection neurons in the poor zones were largely similar to those in the conventional striatum using in vitro electrophysiological recording. In addition, the poor zones existed irrespective of the age or sex of mice. We also identified the poor zones in the common marmoset as well as other rodents. These results suggest that the poor zones in the caudal striatum follow the conventional projection patterns irrespective of the imbalanced distribution of projection neurons. The poor zones could be an innate structure and common in mammals. The unique striatal zones possessing highly restricted projections could relate to functions different from those of motor-related striatum.

Exclusive and common targets of neostriatofugal projections of rat striosome neurons: a single neuron-tracing study using a viral vector.

The rat neostriatum has a mosaic organization composed of striosome/patch compartments embedded in a more extensive matrix compartment, which are distinguished from each other by the input-output organization as well as by the expression of many molecular markers. The matrix compartment gives rise to the dual γ-aminobutyric acid (GABA)ergic striatofugal systems, i.e. direct and indirect pathway neurons, whereas the striosome compartment is considered to involve direct pathway neurons alone. Although the whole axonal arborization of matrix striatofugal neurons has been examined in vivo by intracellular staining, that of striosome neurons has never been studied at the single neuron level. In the present study, the axonal arborizations of single striosome projection neurons in rat neostriatum were visualized in their entirety using a viral vector expressing membrane-targeted green fluorescent protein, and compared with that of matrix projection neurons. We found that not only matrix but also striosome compartments contained direct and indirect pathway neurons. Furthermore, only striatonigral neurons in the striosome compartment projected directly to the substantia nigra pars compacta (SNc), although they sent a substantial number of axon collaterals to the globus pallidus, entopeduncular nucleus and/or substantia nigra pars reticulata. These results suggest that striosome neurons play a more important role in the formation of reward-related signals of SNc dopaminergic neurons than do matrix neurons. Together with data from previous studies in the reinforcement learning theory, our results suggest that these direct and indirect striosome-SNc pathways together with nigrostriatal dopaminergic neurons may help striosome neurons to acquire the state-value function.

Complementary distribution of glutamatergic cerebellar and GABAergic basal ganglia afferents to the rat motor thalamic nuclei.

Motor thalamic nuclei, ventral anterior (VA), ventral lateral (VL) and ventral medial (VM) nuclei, receive massive glutamatergic and GABAergic afferents from the cerebellum and basal ganglia, respectively. In the present study, these afferents were characterized with immunoreactivities for glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular glutamate transporter (VGluT)2, and examined by combining immunocytochemistry with the anterograde axonal labeling and neuronal depletion methods in the rat brain. VGluT2 immunoreactivity was intense in the caudodorsal portion of the VA-VL, whereas GAD67 immunoreactivity was abundant in the VM and rostroventral portion of the VA-VL. The rostroventral VA-VL and VM contained two types of GAD67-immunopositive varicosities (large and small), but the caudodorsal VA-VL comprised small ones alone. VGluT2-immunopositive varicosities were much larger in the caudodorsal VA-VL than those in the rostroventral VA-VL and VM. When anterograde tracers were injected into the basal ganglia output nuclei, the vast majority of labeled axon varicosities were large and distributed in the rostroventral VA-VL and VM, showing immunoreactivity for GAD67, but not for VGluT2. Only the large GAD67-immunopositive varicosities were mostly abolished by kainic acid depletion of substantia nigra neurons. In contrast, large to giant axon varicosities derived from the deep cerebellar nuclei were distributed mostly in the caudodorsal VA-VL, displaying VGluT2 immunoreactivity. The VGluT2-positive varicosities disappeared from the core portion of the caudodorsal VA-VL by depletion of cerebellar nucleus neurons. Thus, complementary distributions of large VGluT2- and GAD67-positive terminals in the motor thalamic nuclei are considered to reflect glutamatergic cerebellar and GABAergic basal ganglia afferents, respectively.

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum.

The axonal arbors of single nigrostriatal dopaminergic neurons were visualized with a viral vector expressing membrane-targeted green fluorescent protein in rat brain. All eight reconstructed tyrosine hydroxylase-positive dopaminergic neurons possessed widely spread and highly dense axonal arborizations in the neostriatum. All of them emitted very little axon collateral arborization outside of the striatum except for tiny arborization in the external pallidum. The striatal axonal bush of each reconstructed dopaminergic neuron covered 0.45-5.7% (mean +/- SD = 2.7 +/- 1.5%) of the total volume of the neostriatum. Furthermore, all the dopaminergic neurons innervated both striosome and matrix compartments of the neostriatum, although each neuron's arborization tended to favor one of these compartments. Our findings demonstrate that individual dopaminergic neurons of the substantia nigra can broadcast a dopamine signal and exert strong influence over a large number of striatal neurons. This divergent signaling should be a key to the function of the nigrostriatal system in dopamine-based learning and suggests that neurodegeneration of individual nigral neurons can affect multiple neurons in the striatum. Thus, these results would also contribute to understanding the clinicopathology of Parkinson's disease and related syndromes.