Corticocortical innervation subtypes of layer 5 intratelencephalic cells in the murine secondary motor cortex.

Feedback projections from the secondary motor cortex (M2) to the primary motor and sensory cortices are essential for behavior selection and sensory perception. Intratelencephalic (IT) cells in layer 5 (L5) contribute feedback projections to diverse cortical areas. Here we show that L5 IT cells participating in feedback connections to layer 1 (L1) exhibit distinct projection patterns, genetic profiles, and electrophysiological properties relative to other L5 IT cells. An analysis of the MouseLight database found that L5 IT cells preferentially targeting L1 project broadly to more cortical regions, including the perirhinal and auditory cortices, and innervate a larger volume of striatum than the other L5 IT cells. We found experimentally that in upper L5 (L5a), ER81 (ETV1) was found more often in L1-preferring IT cells, and in IT cells projecting to perirhinal/auditory regions than those projecting to primary motor or somatosensory regions. The perirhinal region-projecting L5a IT cells were synaptically connected to each other and displayed lower input resistance than contra-M2 projecting IT cells including L1-preferring and nonpreferring cells. Our findings suggest that M2-L5a IT L1-preferring cells exhibit stronger ER81 expression and broader cortical/striatal projection fields than do cells that do not preferentially target L1.

Ipsi- and contralateral corticocortical projection-dependent subcircuits in layer 2 of the rat frontal cortex.

In the neocortex, both layer 2/3 and layer 5 contain corticocortical pyramidal cells projecting to other cortices. We previously found that among L5 pyramidal cells of the secondary motor cortex (M2), not only intratelencephalic projection cells but also pyramidal tract cells innervate ipsilateral cortices and that the two subtypes are different in corticocortical projection diversity and axonal laminar distributions. Layer 2/3 houses intratelencephalically projecting pyramidal cells that also innervate multiple ipsilateral and contralateral cortices. However, it remained unclear whether layer 2/3 pyramidal cells can be divided into projection subtypes each with distinct innervation to specific targets. In the present study we show that layer 2 pyramidal cells are organized into subcircuits on the basis of corticocortical projection targets. Layer 2 corticocortical cells of the same projection subtype were monosynaptically connected. Between the contralaterally and ipsilaterally projecting corticocortical cells, the monosynaptic connection was more common from the former to the latter. We also found that ipsilaterally and contralaterally projecting corticocortical cell subtypes differed in their morphological and physiological characteristics. Our results suggest that layer 2 transfers separate outputs from M2 to individual cortices and that its subcircuits are hierarchically organized to form the discrete corticocortical outputs.NEW & NOTEWORTHY Pyramidal cell subtypes and their dependent subcircuits are well characterized in cortical layer 5, but much less is understood for layer 2/3. We demonstrate that in layer 2 of the rat secondary motor cortex, ipsilaterally and contralaterally projecting corticocortical cells are largely segregated. These layer 2 cell subtypes differ in dendrite morphological and intrinsic electrophysiological properties, and form subtype-dependent connections. Our results suggest that layer 2 pyramidal cells form distinct subcircuits to provide discrete corticocortical outputs.

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.

Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices.

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.

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). PRC-projecting cells in upper layer 2/3 (L2/3) of the frontal cortex projected to perirhinal area 35, while neurons in L5 innervated perirhinal area 36. L2/3 PRC-projecting cells partially overlapped with those projecting to the basolateral amygdala. L5 PRC-projecting cells partially overlapped with crossed corticostriatal cells, but were distinct from neighboring corticothalamic (CTh)/corticopontine cells. L5 PRC-projecting and CTh cells were different in their electrophysiological properties and dendritic/axonal morphologies. Within the frontal cortex, L2/3 PRC-projecting cells innervated L5 PRC-projecting and CTh cells with similar probabilities, but received feedback excitation only from PRC-projecting cells. These data suggest that specific neuron subtypes in different cortical layers are reciprocally excited via interlaminar loops. Thus, two interacting output channels send information from the frontal cortex to different hierarchical stages of the parahippocampal network, areas 35 and 36, with additional collaterals selectively targeting the amygdala or basal ganglia, respectively. Combined with the hierarchical connectivity of PRC-projecting and CTh cells, these observations demonstrate an exquisite diversification of frontal projection neurons selectively connected according to their participation in distinct memory subsystems.

High resolution recording of local field currents simultaneously with sound-evoked calcium signals by a photometric patch electrode in the auditory cortex field L of the chick.

BACKGROUND: Functioning of the brain is based on both electrical and metabolic activity of neural ensembles. Accordingly, it would be useful to measure intracellular metabolic signaling simultaneously with electrical activity in the brain in vivo. NEW METHOD: We innovated a PhotoMetric-patch-Electrode (PME) recording system that has a high temporal resolution incorporating a photomultiplier tube as a light detector. The PME is fabricated from a quartz glass capillary to transmit light as a light guide, and it can detect electrical signals as a patch electrode simultaneously with a fluorescence signal. RESULTS: We measured the sound-evoked Local Field Current (LFC) and fluorescence Ca2+ signal from neurons labeled with Ca2+-sensitive dye Oregon Green BAPTA1 in field L, the avian auditory cortex. Sound stimulation evoked multi-unit spike bursts and Ca2+ signals, and enhanced the fluctuation of LFC. After a brief sound stimulation, the cross-correlation between LFC and Ca2+ signal was prolonged. D-AP5 (antagonist for NMDA receptors) suppressed the sound-evoked Ca2+ signal when applied locally by pressure from the tip of PME. COMPARISON WITH EXISTING METHODS: In contrast to existing multiphoton imaging or optical fiber recording methods, the PME is a patch electrode pulled simply from a quartz glass capillary and can measure fluorescence signals at the tip simultaneously with electrical signal at any depth of the brain structure. CONCLUSION: The PME is devised to record electrical and optical signals simultaneously with high temporal resolution. Moreover, it can inject chemical agents dissolved in the tip-filling medium locally by pressure, allowing manipulation of neural activity pharmacologically.

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. We further correlated morphological and physiological characteristics of identified some chemical subclasses of inhibitory neurons. Our results reveal expression specificity of CRF, NPY, SPR, and NOS in morphologically and physiologically distinct interneuron classes. These observations support the existence of a limited number of functionally distinct subtypes of GABA cells in the neocortex.

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.

Quantitative chemical composition of cortical GABAergic neurons revealed in transgenic venus-expressing rats.

Although neocortical GABAergic (gamma-aminobutyric acidergic) interneurons have been the focus of intense study, especially in the rat, a consensus view of the functional diversity and organization of inhibitory cortical neurons has not yet been achieved. To better analyze GABAergic neurons in the rat, we used a bacterial artificial chromosome (BAC) construct and established 2 lines of transgenic rats that coexpress Venus, a yellow fluorescent protein, with the vesicular GABA transporter. The brain GABA content from both transgenic lines was similar to the level found in wild-type rats. In the frontal cortex, Venus was expressed in >95% of GABAergic neurons, most of which also expressed at least one of 6 biochemical markers, including alpha-actitin-2, which preferentially labeled late-spiking neurogliaform cells. Taking advantage of the fact that Venus expression allows for targeted recording from all classes of nonpyramidal cells, irrespective of their somatic morphologies, we demonstrated that fast-spiking neurons, which were heterogeneous in somatic size as well as vertical dendritic projection, had relatively uniform horizontal dimensions, suggesting a cell type-specific columnar input territory. Our data demonstrate the benefits of VGAT-Venus rats for investigating GABAergic circuits, as well as the feasibility of using BAC technology in rats to label subsets of specific, genetically defined neurons.

Direction- and distance-dependent interareal connectivity of pyramidal cell subpopulations in the rat frontal cortex.

The frontal cortex plays an important role in the initiation and execution of movements via widespread projections to various cortical and subcortical areas. Layer 2/3 (L2/3) pyramidal cells in the frontal cortex send axons mainly to other ipsilateral/contralateral cortical areas. Subpopulations of layer 5 (L5) pyramidal cells that selectively project to the pontine nuclei or to the contralateral cortex [commissural (COM) cells] also target diverse and sometimes overlapping ipsilateral cortical areas. However, little is known about target area-dependent participation in ipsilateral corticocortical (iCC) connections by subclasses of L2/3 and L5 projection neurons. To better understand the functional hierarchy between cortical areas, we compared iCC connectivity between the secondary motor cortex (M2) and adjacent areas, such as the orbitofrontal and primary motor cortices, and distant non-frontal areas, such as the perirhinal and posterior parietal cortices. We particularly assessed the laminar distribution of iCC cells and fibers, and identified the subtypes of pyramidal cells participating in those projections. For connections between M2 and frontal areas, L2/3 and L5 cells in both areas contributed to reciprocal projections, which can be viewed as "bottom-up" or "top-down" on the basis of their differential targeting of cortical lamina. In connections between M2 and non-frontal areas, neurons participating in bottom-up and top-down projections were segregated into the different layers: bottom-up projections arose primarily from L2/3 cells, while top-down projections were dominated by L5 COM cells. These findings suggest that selective participation in iCC connections by pyramidal cell subtypes lead to directional connectivity between M2 and other cortical areas. Based on these findings, we propose a provisional unified framework of interareal hierarchy within the frontal cortex, and discuss the interaction of local circuits with long-range interareal connections.

High-energy phase-contrast X-ray imaging using a two-crystal X-ray interferometer.

To broaden the scope of phase-contrast X-ray imaging in biomedical applications, the energy of X-rays in a large-area imaging system using a two-crystal X-ray interferometer has been increased from 17.7 keV to 35 keV. The use of higher-energy X-rays makes it possible to observe larger samples having greater variation in density within shorter measurement periods, at higher spatial resolution, and at lower X-ray doses. After optimizing the imaging system for 35 keV X-rays, a 25 mm x 30 mm interference pattern with 50% visibility was generated at the Photon Factory. The system's capabilities are demonstrated by this pattern and a high-quality three-dimensional image of a rat heart.