Icing after skeletal muscle injury with necrosis in a small fraction of myofibers limits inducible nitric oxide synthase-expressing macrophage invasion and facilitates muscle regeneration.

Growing evidence from animal experiments suggests that icing after skeletal muscle injury is harmful to muscle regeneration. However, these previous experimental models yielded massive necrotic myofibers, whereas muscle injury with necrosis in a small myofiber fraction (<10%) frequently occurs in human sports activities. Although macrophages play a proreparative role during muscle regeneration, they exert a cytotoxic effect on muscle cells through an inducible nitric oxide synthase (iNOS)-mediated mechanism. In this study, we established an animal injury model with necrosis limited to a small myofiber fraction and investigated the effect of icing on muscle regeneration with a focus on macrophage-related events. Icing after muscle injury of this model resulted in an enlarged size of regenerating myofibers compared with those in untreated animals. During the regenerative process, icing attenuated the accumulation of iNOS-expressing macrophages, suppressed iNOS expression in the whole damaged muscle, and limited the expansion of the injured myofiber area. In addition, icing increased the ratio of M2 macrophages within the injured site at an earlier time point than that in untreated animals. Following these phenomena in icing-treated muscle regeneration, an early accumulation of activated satellite cells within the damaged/regenerating area occurred. The expression level of myogenic regulatory factors, such as MyoD and myogenin, was not affected by icing. Taken together, our results suggest that icing after muscle injury with necrosis limited to a small fraction of myofibers facilitates muscle regeneration by attenuating iNOS-expressing macrophage invasion, limiting muscle damage expansion, and accelerating the accumulation of myogenic cells which form regenerating myofibers.

Comparison of the soleus and plantaris muscles in humans and other primates: Macroscopic neuromuscular anatomy and evolutionary significance.

In humans, the soleus is more developed compared to other primates and has a unique architecture composed of anterior bipennate and posterior unipennate parts, which are innervated by different nerve branches. The anterior part of the human soleus was proposed to be important for bipedalism, however, the phylogenetic process resulting in its acquisition remains unclear. Providing insights into this process, the anterior part of the soleus was suggested to be closely related to the plantaris based on the branching pattern of their nerve fascicles. To reveal the phylogeny of the soleus and plantaris in primates, the innervation patterns of the posterior crural muscles were compared among a wide range of species. From their branching pattern, posterior crural muscles could be classified into superficial and deep muscle groups. The anterior part of the soleus and plantaris both belonged to the deep muscle group. In all the examined specimens of ring-tailed lemurs and chimpanzees, as well as in one out of two specimens of siamang, the nerve branches corresponding to those innervating the anterior part of the human soleus were found. The muscular branches innervating the anterior part of the soleus and plantaris formed a common trunk or were connected in all the specimens. These results indicate that the anterior part of the soleus is closely related to the plantaris across different species of primates. In turn, this suggests that the anterior part of the soleus is maintained among primates, and especially in humans, where it develops as the characteristic bipennate structure.

Three-dimensional anatomical structure formed by granule cell layer and pyramidal cell layer in human hippocampus.

In the human hippocampus, the pyramidal layer consists of the inferior aspect of the hippocampus which is organized segmentally. Each segment, together with granule layer of the dentate gyrus, exhibits structural unity. In humans, ellipsoidal protrusions called pyramidal hillocks (PHs), which consist of a thick pyramidal cell layer (PL), are present in the inferior aspect of the hippocampus, and are segmentally organized along a longitudinal axis. It is also known that the granule cell layer (GL) of the dentate gyrus (DG) is not a smooth but undulated structure. However, the cytoarchitectural relationships between the protrusions and undulation have yet to be studied well. Here, we aimed to clarify the three-dimensional cytoarchitecture of the PL and GL of human hippocampus. For that purpose, the GL and PL were three-dimensionally reconstructed from serial sections of human hippocampus stained with hematoxylin and eosin. The GL was shaped as tubing with an opening in the dorsal part, and undulated especially in the medial part, forming digit-like processes. In the base of a digit-like process, protrusions of the GL extended laterally, with longer ones reaching the lateral edge, whereas shorter ones disappeared around the medial 1/3 of the GL. Consequently, the lateral part of the GL was undulated loosely. In the ventral view of the PL, the ellipsoidal PHs were sagittally aligned, whereas in the top view, each PH formed an ellipsoidal trough. Each structural unit was formed by a trough of the PH along the bottom, and had a longer GL protrusion in the upper-center, and shorter GL protrusions located between the longer protrusions and the lateral edge of the GL. A digit-like process extended into a dens. It is concluded that a unit of the PH and the GL comprises the longitudinal segmental formation of the hippocampus.

Icing after skeletal muscle injury decreases M1 macrophage accumulation and TNF-α expression during the early phase of muscle regeneration in rats.

Following skeletal muscle injury, both myogenic and immune cells interact closely during the regenerative process. Although icing is still a common acute treatment for sports-related skeletal muscle injuries, icing after muscle injury has been shown to disrupt macrophage accumulation and impair muscle regeneration in animal models. However, it remains unknown whether icing shortly after injury affects macrophage-related phenomena during the early stages of muscle regeneration. Therefore, we focused on the distribution of M1/M2 macrophages and cytokines expressed predominantly by macrophages during the early stages of muscle regeneration after muscle crush injury. Icing resulted in a decrease, not retardation, in the accumulation of M1 macrophages, but not M2 macrophages, in injured muscles. Consistent with the decrease in M1 macrophage accumulation, icing led to a reduction, instead of delay, in the level of tumor necrosis factor-α (TNF-α) expression. Additionally, at subsequent timepoints, icing decreased the number of myogenic precursor cells in the regenerating area and the size of centrally nucleated regenerating myofibers. Together, our findings suggest that icing after acute muscle damage by crushing disturbs muscle regeneration through hindering tM1 macrophage-related phenomena.

Icing after eccentric contraction-induced muscle damage perturbs the disappearance of necrotic muscle fibers and phenotypic dynamics of macrophages in mice.

Icing is still one of the most common treatments to acute skeletal muscle damage in sports medicine. However, previous studies using rodents reported the detrimental effect of icing on muscle regeneration following injury. This study aimed to elucidate the critical factors governing the impairment of muscle regeneration by icing with a murine model of eccentric contraction-induced muscle damage by electrical stimulation. Because of icing after muscle injury, the infiltration of polynuclear and mononuclear cells into necrotic muscle fibers was retarded and attenuated, leading to the persistent presence of necrotic cellular debris. These phenomena coincided with the delayed emergence and sustained accumulation of Pax7+ myogenic cells within the regenerating area. In addition, due to icing, delayed and/or sustained infiltration of M1 macrophages was noted in accordance with the perturbed expression patterns of inflammation-related factors, including tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). The key myogenic regulatory factors (i.e., MyoD and myogenin) involved in the activation/proliferation and differentiation of myogenic precursor cells were not altered by icing during the regenerative process. A detailed analysis of regenerating myofibers by size distribution at day 14 after muscle damage showed that the ratio of small regenerating fibers to total regenerating fibers was higher in icing-treated animals than in untreated animals. These findings suggest that icing following muscle damage blunts the efficiency of muscle regeneration by perturbing the removal of necrotic myofibers and phenotypic dynamics of macrophages rather than affecting myogenic factors.NEW & NOTEWORTHY Icing blunted the muscle regeneration by perturbing the infiltration of polynuclear and mononuclear cells into necrotic myofibers and the phenotypic dynamics of macrophages rather than affecting the myogenic regulatory factors. Because of icing, the disappearance of necrotic muscle debris was retarded, coinciding with the delayed emergence and sustained accumulation of Pax7+ cells within the regenerating area. The expression patterns of TNF-α and IL-10 were altered by icing consistent with the perturbation of the macrophage phenotype.

Topographical relationship between the accessory hepatic duct and the hepatic artery system.

Hepatic biliary injury is one of the most common complications in cholecystectomy and is frequently accompanied by arterial injuries. Because there are several anatomical variations of the hepatic ducts, including the accessory hepatic ducts (AHDs), it is important to consider not only the anatomical position of the hepatic ducts but also those of the AHDs in cholecystectomy. However, the topographical relationships between the AHDs and the hepatic arteries are still poorly understood. In the present study we show that AHDs were observed in 7 out of 59 (11.9%) of the cadavers. There was a single AHD in the 6 out of the 7 cadavers and double AHDs in one. In these cases, the right AHDs emerged from the anterior medial segment of the liver piercing the parenchyma, while the left AHDs emerged directly from the anterior part of the caudate lobe. The right AHDs ran anterior to the right hepatic artery, while the left AHDs ran posterior to the hepatic arteries. The topographical relationship between the AHD and the hepatic artery system was thus reversed in the cases of the right and the left AHDs.

Anatomical variations of the torcular Herophili: macroscopic study and clinical aspects.

The anatomical variations of the confluence of sinuses were examined, focusing on the continuity of the superior sagittal sinus (SSS) and the transverse sinuses (TSs). In the 142 specimens studied, there were 72 symmetric cases (50.7%) and 70 asymmetric cases (49.3%). The symmetric group (no dominant type) was categorized into 34 cases of bifurcation (23.9%) and 38 cases of confluence (26.8%). The asymmetric group was categorized into 54 cases of the right-dominant type (38.0%) and 16 cases of the left-dominant type (11.3%). The right-dominant type was further categorized into 38 partially-communicating (26.8%) and 16 non-communicating types (11.3%). The left-dominant type was categorized into 11 partially-communicating (7.7%) and 5 non-communicating types (3.5%). In summary, the SSS asymmetrically drained into one TS in about half of the cases studied. The right-dominant type was about three to four times as common as the left-dominant type. The draining pattern shown by the asymmetric group could provoke intracranial hypertension due to unilateral jugular vein obstruction. In order to avoid this risk in cases of neck dissection, jugular vein catheterization, or hypercoagulopathy, preoperative evaluations of the dural sinus variations via MR venography, three-dimensional CT, or plain X-ray of the skull are recommended.

The middle rectal artery arising from the lateral sacral artery.

A middle rectal artery arising from the lateral sacral artery (MRAls) in the right pelvis of a 99-year-old male was observed. Although variations of the origin of the middle rectal artery have been reported on many occasions, there are few descriptions of the trajectory in the literature. In our case, the MRAls branched from the lateral sacral artery on the sacral surface close to the third sacral sympathetic ganglion and immediately penetrated the third sacral splanchnic nerve and the parasympathetic pelvic splanchnic nerve from the ventral ramus of the forth sacral nerve. The MRAls entered in the lateral wall of the rectal ampulla without giving off a prostatic branch. Preservation of the pelvic autonomic nerves are crucial in rectal cancer excision to preserve the autonomic functions. The close topography of the MRAls to the origin of the fine autonomic nerves should be noted.

Anatomical variations of the recurrent artery of Heubner: number, origin, and course.

The clinical anatomy of the recurrent artery of Heubner (RAH) was examined, focusing on its number, origin, and course, in a large number of brain specimens. We studied 724 RAH in total from 357 brain specimens (714 hemispheres). In 98.74 % of 714 cases there were one or more RAHs, while it was absent in 1.26 % of cases. There was a single RAH in 96.22 % of cases, double in 2.38 % of cases, and triple in 0.14 % of cases. In this study, three origin types of the RAH were defined. We defined A1 and A2 segment of the anterior cerebral artery (ACA) as the artery from the origin of the ACA to the junction of the anterior communicating artery (AComA) and the artery from the junction of the AComA to the anterior border of the corpus callosum, respectively. In 76.2 % of 724 arteries, the RAH originated from the junction of the A1 and A2 segment of the ACA. In 16.3 %, the RAH originated from the A2 segment of the ACA. In 7.5 %, the RAH originated from the A1 segment of the ACA. The course of the RAH was superior to the A1 segment of the ACA in 30.1 % of 724 arteries, anterior in 62.2 %, and posterior in 7.7 %. It is of great importance for neurosurgeons to understand the detailed anatomical variations of the RAH before operating to prevent operative complications resulting in neurological deficits.

Neonatal maternal separation delays the GABA excitatory-to-inhibitory functional switch by inhibiting KCC2 expression.

The excitatory-to-inhibitory functional switch of γ-aminobutyric acid (GABA; GABA switch), which normally occurs in the first to the second postnatal week in the hippocampus, is necessary for the development of appropriate central nervous system function. A deficit in GABAergic inhibitory function could cause excitatory/inhibitory (E/I) neuron imbalance that is found in many neurodegenerative disorders. In the present study, we examined whether neonatal stress can affect the timing of the GABA functional switch and cause disorders during adolescence. Neonatal stress was induced in C57BL/6J male mouse pups by maternal separation (MS) on postnatal days (PND) 1-21. Histological quantification of K+-Cl- co-transporter (KCC2) and Ca2+ imaging were performed to examine the timing of the GABA switch during the MS period. To evaluate the influence of neonatal MS on adolescent hippocampal function, we quantified KCC2 expression and evaluated hippocampal-related behavioral tasks at PND35-38. We showed that MS delayed the timing of the GABA switch in the hippocampus and inhibited the increase in membrane KCC2 expression, with KCC2 expression inhibition persisting until adolescence. Behavioral tests showed impaired cognition, declined attention, hyperlocomotion, and aggressive character in maternally separated mice. Taken together, our results show that neonatal stress delayed the timing of the GABA switch, which could change the E/I balance and cause neurodegenerative disorders in later life.

Three-dimensional analysis of seminiferous tubules and spermatogenic waves in mice.

The aim of the present study was to reconstruct seminiferous tubules and analyze spermatogenic waves in seminiferous epithelia in developing and adult mice using serial paraffin sections and high-performance three-dimensional (3D) reconstruction software. By labeling the basement membrane of seminiferous tubules with fluorescent immunohistochemistry or periodic acid-Schiff-hematoxylin staining, all seminiferous tubules were reconstructed in 9 testes from 9 different mice, 3 each at 0, 21 and 90 days (adult) postpartum. The 3D structure of seminiferous tubules, including the number and length of tubules as well as the number of connections with the rete testis, branching points and blind ends, was assessed accurately. Although tubules showed marked variations among individual mice, their overall structure was regular and retained from newborn to adult mice. Some seminiferous tubules contained inner portions running distant from the testis surface. In a representative testis at 21 days, the sites at which spermatids initially occurred were examined by labeling acrosomes and were found to be preferentially distributed in the upper and medial portions of the testis close to the rete testis. In a representative adult testis, 76 complete waves with an average length of 16.9 mm were found and their directions were analyzed. The methods used in the present study will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions, such as infertility.

Clinical anatomy of the cephalic vein for safe performance of venipuncture.

BACKGROUND: The aims of this study were to elucidate why the cephalic vein provides a reliable cannulation site from a morphological viewpoint and identify an effective landmark for avoiding injury to the superficial branch of the radial nerve (SBRN), allowing for safe venipuncture of the cephalic vein. FINDINGS: We examined 32 forearms and wrists from 18 cadavers. The cephalic vein was a constant structure containing a branch communicating with a collateral vein of the deep palmar arch via the first dorsal interossei muscle. The metacarpal vein from the medial two digits flowed into the cephalic vein. The venous confluence formed 5.8 ± 1.2 cm proximal to the radial styloid process. The SBRN passed 0.4 ± 0.3 cm volar to the venous confluence. The distance between the venous confluence and subcutaneous emergence of the SBRN was 2.6 ± 1.0 cm. CONCLUSIONS: These observations suggest that the cephalic vein is a constant structure that serves as a drainage vein of the hand and provides a reliable cannulation site in the forearm. The venous confluence may serve as a novel landmark to predict the running course of the SBRN.

The effect of repeated stress on KCC2 and NKCC1 immunoreactivity in the hippocampus of female mice.

K(+)-Cl(-) co-transporter (KCC2) and Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) are the main regulators of neuronal intracellular chloride concentration; altered expression patterns of KCC2 and NKCC1 have been reported in several neurodegenerative diseases. In this paper, we show the effect of repeated stress on KCC2, NKCC1, and serine 940 phosphorylated KCC2 (pKCC2(ser940)) immunoreactivity. The data were obtained from the hippocampus of female mice using single-plane confocal microscopy images. The mean fluorescence intensity of the perisomatic area of neurons, defined as raw fluorescence intensity (RFI) was calculated. Repeated stress (RS) resulted in a decrease in perisomatic area of immunoreactive (IR)-KCC2 and an increase of the IR-NKCC1. In addition, RS decreased perisomatic IR-pKCC2(ser940), corresponding to that of KCC2. The data in this article support the results of a previous study [1] and provide the details of immunohistological methods. Interpretation of the data in this article can be found in "Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice" by Tsukahara et al. [1].

Three-dimensional structure of seminiferous tubules in the adult mouse.

Seminiferous tubules develop from sex cords, which are embryonic structures with simple C-shaped arches. Histologically, the epithelium of adult mouse seminiferous tubules has been divided into 12 stages based on the associations of spermatogenic cells in four cycles of spermatogenesis. However, the gross characteristics of the seminiferous tubules themselves, including their number, length, run, and mutual relationships remain largely unknown. In the present study, we analyzed all seminiferous tubules in a single adult mouse testis with high resolution using serial paraffin sections and high-perfomance three-dimensional reconstruction software. There were 11 seminiferous tubules with an average length of 140 mm. Each tubule ran along circular paths within the testis while making convolutions with cranial and caudal hairpin turns. The cranial turns of all tubules were in contact with the tunica albuginea, whereas the caudal turns were not, resulting in funnel-shaped networks of these tubules with tapered caudal portions. The caudally located networks surrounded the preceding cranially located networks from the bottom and outside, similar to stacked paper cups. Five out of the 11 seminiferous tubules were continuous from one end to the other both connected with the rete testis (10 connection points). Nine branching points, one blind end, and 18 more connection points with the rete testis were detected in the remaining six seminiferous tubules, making the paths of these tubules complicated to various degrees. The present study revealed that the 3D structures of seminiferous tubules were highly regular as a whole in the adult mouse testis.

Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice.

The balance of cation-chloride co-transporters, particularly KCC2 and NKCC1, is critical for GABAergic inhibitory signaling. However, KCC2/NKCC1 balance is disrupted in many neurodegenerative diseases. Moreover, correlations between chronic stress, KCC2 and NKCC1 in the hippocampus remain poorly understood. Despite the fact that emotional disorders in humans are far more prevalent in women, there have been relatively few studies about female subjects. Here we investigated behaviors and expression patterns of KCC2 and NKCC1 in the hippocampi of female mice under chronic stress. Repeated stress (RS) was induced in experimental mice by repeated forced water administration. Then, expression patterns of GABAergic signaling molecules were identified by immunohistochemical analysis and performance was assessed using several behavioral tests. The results of semi-quantitative analysis showed that RS decreased KCC2 expression and increased NKCC1 expression in membranes of granular and pyramidal cells in the hippocampus. The novel object recognition (NOR) test and sociability test revealed that RS induced cognitive and sociability deficits, whereas RS increased the time spent in the open arms of the elevated plus maze test and induced attention deficits in other tests. In summary, RS induced alterations in membrane KCC2/NKCC1 balance in the hippocampus of female mice, which may contribute to GABAergic disinhibition associated with cognitional, sociability and attention deficits.

Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM.

Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.

Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

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.

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.

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.