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.

descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner.

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.

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.

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.

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.

Modulatory Effects of Monoamines and Perineuronal Nets on Output of Cerebellar Purkinje Cells.

Classically, the cerebellum has been thought to play a significant role in motor coordination. However, a growing body of evidence for novel neural connections between the cerebellum and various brain regions indicates that the cerebellum also contributes to other brain functions implicated in reward, language, and social behavior. Cerebellar Purkinje cells (PCs) make inhibitory GABAergic synapses with their target neurons: other PCs and Lugaro/globular cells via PC axon collaterals, and neurons in the deep cerebellar nuclei (DCN) via PC primary axons.

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).

Motor cortex can directly drive the globus pallidus neurons in a projection neuron type-dependent manner in the rat.

The basal ganglia are critical for the control of motor behaviors and for reinforcement learning. Here, we demonstrate in rats that primary and secondary motor areas (M1 and M2) make functional synaptic connections in the globus pallidus (GP), not usually thought of as an input site of the basal ganglia. Morphological observation revealed that the density of axonal boutons from motor cortices in the GP was 47% and 78% of that in the subthalamic nucleus (STN) from M1 and M2, respectively.

Thalamostriatal projections and striosome-matrix compartments.

The neostriatum has a mosaic organization consisting of striosome and matrix compartments. It receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Recent behavioral studies in rats revealed a selectively active medial prefronto-striosomal circuit during cost-benefit decision-making.

Supramammillary Nucleus Afferents to the Dentate Gyrus Co-release Glutamate and GABA and Potentiate Granule Cell Output.

The supramammillary nucleus (SuM) of the hypothalamus projects to the dentate gyrus (DG) and the CA2 region of the hippocampus. Although the SuM-to-hippocampus circuits have been implicated in spatial and emotional memory formation, little is known about precise neural connections between the SuM and hippocampus. Here, we report that axons of SuM neurons make monosynaptic connections to granule cells (GCs) and GABAergic interneurons, but not to hilar mossy cells, in the DG and co-release glutamate and γ-aminobutyric acid (GABA) at these synapses.

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).

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.

Substance P effects exclusively on prototypic neurons in mouse globus pallidus.

Previous studies have suggested that the neurokinin-1 receptor (NK-1R) expressing neurons in the globus pallidus (GP) receive substance P (SP), presumably released by axon collaterals of striatal direct neurons. However, the effect of SP on the GP remains unclear. In this study, we identified that the SP-responsive cells comprise a highly specific cell type in the GP with regard to immunofluorescence, electrophysiology, and projection properties.

Using a novel PV-Cre rat model to characterize pallidonigral cells and their terminations.

In the present study, we generated a novel parvalbumin (PV)-Cre rat model and conducted detailed morphological and electrophysiological investigations of axons from PV neurons in globus pallidus (GP). The GP is considered as a relay nucleus in the indirect pathway of the basal ganglia (BG). Previous studies have used molecular profiling and projection patterns to demonstrate cellular heterogeneity in the GP; for example, PV-expressing neurons are known to comprise approximately 50% of GP neurons and represent majority of prototypic neurons that project to the subthalamic nucleus and/or output nuclei of BG, entopeduncular nucleus and substantia nigra (SN).

Axon topography of layer 6 spiny cells to orientation map in the primary visual cortex of the cat (area 18).

To uncover the functional topography of layer 6 neurons, optical imaging was combined with three-dimensional neuronal reconstruction. Apical dendrite morphology of 23 neurons revealed three distinct types. Type Aa possessed a short apical dendrite with many oblique branches, Type Ab was characterized by a short and less branched apical dendrite, whereas Type B had a long apical dendrite with tufts in layer 2.

The Diversity of Cortical Inhibitory Synapses.

The most typical and well known inhibitory action in the cortical microcircuit is a strong inhibition on the target neuron by axo-somatic synapses. However, it has become clear that synaptic inhibition in the cortex is much more diverse and complicated. Firstly, at least ten or more inhibitory non-pyramidal cell subtypes engage in diverse inhibitory functions to produce the elaborate activity characteristic of the different cortical states.

Neuronal circuits and physiological roles of the basal ganglia in terms of transmitters, receptors and related disorders.

The authors have reviewed recent research advances in basal ganglia circuitry and function, as well as in related disorders from multidisciplinary perspectives derived from the results of morphological, electrophysiological, behavioral, biochemical and molecular biological studies. Based on their expertise in their respective fields, as denoted in the text, the authors discuss five distinct research topics, as follows: (1) area-specific dopamine receptor expression of astrocytes in basal ganglia, (2) the role of physiologically released dopamine in the striatum, (3) control of behavioral flexibility by striatal cholinergic interneurons, (4) regulation of phosphorylation states of DARPP-32 by protein phosphatases and (5) physiological perspective on deep brain stimulation with optogenetics and closed-loop control for ameliorating parkinsonism.

Functional effects of distinct innervation styles of pyramidal cells by fast spiking cortical interneurons.

Inhibitory interneurons target precise membrane regions on pyramidal cells, but differences in their functional effects on somata, dendrites and spines remain unclear. We analyzed inhibitory synaptic events induced by cortical, fast-spiking (FS) basket cells which innervate dendritic shafts and spines as well as pyramidal cell somata. Serial electron micrograph (EMg) reconstructions showed that somatic synapses were larger than dendritic contacts.

Morphological elucidation of basal ganglia circuits contributing reward prediction.

Electrophysiological studies in monkeys have shown that dopaminergic neurons respond to the reward prediction error. In addition, striatal neurons alter their responsiveness to cortical or thalamic inputs in response to the dopamine signal, via the mechanism of dopamine-regulated synaptic plasticity. These findings have led to the hypothesis that the striatum exhibits synaptic plasticity under the influence of the reward prediction error and conduct reinforcement learning throughout the basal ganglia circuits.

Long-lasting single-neuron labeling by in vivo electroporation without microscopic guidance.

In order to make a direct link between the morphological and functional study of the nervous system, we established an experimental protocol for labeling individual neurons persistently without microscopic guidance by injecting a plasmid encoding fluorescent protein electroporatively after recording their activity extracellularly. Using a glass pipette filled with electrolyte solution containing a plasmid encoding green fluorescent protein (GFP), single-neuron recording and electroporation were performed on anesthetized rats. When performing the electroporation at the completion of recording, the degree of contact between the target neuron and the electrode tip was adjusted by monitoring the change of the trace of recorded action potentials and the increase of electrode resistance.

Conserved properties of dendritic trees in four cortical interneuron subtypes.

Dendritic trees influence synaptic integration and neuronal excitability, yet appear to develop in rather arbitrary patterns. Using electron microscopy and serial reconstructions, we analyzed the dendritic trees of four morphologically distinct neocortical interneuron subtypes to reveal two underlying organizational principles common to all. First, cross-sectional areas at any given point within a dendrite were proportional to the summed length of all dendritic segments distal to that point.

Specialized cortical subnetworks differentially connect frontal cortex to parahippocampal areas.

How information is manipulated and segregated within local circuits in the frontal cortex remains mysterious, in part because of inadequate knowledge regarding the connectivity of diverse pyramidal cell subtypes. The frontal cortex participates in the formation and retrieval of declarative memories through projections to the perirhinal cortex, and in procedural learning through projections to the striatum/pontine nuclei. In rat frontal cortex, we identified two pyramidal cell subtypes selectively projecting to distinct subregions of perirhinal cortex (PRC).

Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons.

Whether neocortical γ-aminobutyric acid (GABA) cells are composed of a limited number of distinct classes of neuron, or whether they are continuously differentiated with much higher diversity, remains a contentious issue for the field. Most GABA cells of rat frontal cortex have at least 1 of 6 chemical markers (parvalbumin, calretinin, alpha-actinin-2, somatostatin, vasoactive intestinal polypeptide, and cholecystokinin), with each chemical class comprising several distinct neuronal subtypes having specific physiological and morphological characteristics. To better clarify GABAergic neuron diversity, we assessed the colocalization of these 6 chemical markers with corticotropin-releasing factor (CRF), neuropeptide Y (NPY), the substance P receptor (SPR), and nitric oxide synthase (NOS); these 4 additional chemical markers suggested to be expressed diversely or specifically among cortical GABA cells.