Yasuyo Tanaka

Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells

Adult mammalian neurogenesis occurs in the hippocampus and the olfactory bulb, whereas neocortical adult neurogenesis remains controversial. Several occurrences of neocortical adult neurogenesis in injured neocortex were recently reported, suggesting that neural stem cells (NSCs) or neuronal progenitor cells (NPCs) that can be activated by injury are maintained in the adult brain. However, it is not clear whether or where neocortical NSCs/NPCs exist in the brain. We found NPCs in the neocortical layer 1 of adult rats and observed that their proliferation was highly activated by global forebrain ischemia. Using retrovirus-mediated labeling of layer 1 proliferating cells with membrane-targeted green fluorescent protein, we found that the newly generated neurons were GABAergic and that the neurons were functionally integrated into the neuronal circuitry. Our results suggest that layer 1 NPCs are a source of adult neurogenesis under ischemic conditions.

Transiently increased colocalization of vesicular glutamate transporters 1 and 2 at single axon terminals during postnatal development of mouse neocortex: a quantitative analysis with correlation coefficient

Vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 show complementary distribution in neocortex; VGLUT1 is expressed mainly in axon terminals of neocortical neurons, whereas VGLUT2 is located chiefly in thalamocortical axon terminals. However, we recently reported a frequent colocalization of VGLUT1 and VGLUT2 at a subset of axon terminals in postnatal developing neocortex. We here quantified the frequency of colocalization between VGLUT1 and VGLUT2 immunoreactivities at single axon terminals by using the correlation coefficient (CC) as an indicator in order to determine the time course and spatial extent of the colocalization during postnatal development of mouse neocortex. The colocalization was more frequent in the primary somatosensory (S1) area than in both the primary visual (V1) and the motor areas; of area S1 cortical layers, colocalization was most evident in layer IV barrels at postnatal day (P) 7 and in adulthood. CC in layer IV showed a peak at P7 in area S1, and at P10 in area V1 though the latter peak was much smaller than the former. These results suggest that thalamocortical axon terminals contained not only VGLUT2 but also VGLUT1, especially at P7–10. Double fluorescence in situ hybridization confirmed coexpression of VGLUT1 and VGLUT2 mRNAs at P7 in the somatosensory thalamic nuclei and later in the thalamic dorsal lateral geniculate nucleus. As VGLUT1 is often used in axon terminals that show synaptic plasticity in adult brain, the present findings suggest that VGLUT1 is used in thalamocortical axons transiently during the postnatal period when plasticity is required.

Two distinct layer-specific dynamics of cortical ensembles during learning of a motor task

The primary motor cortex (M1) possesses two intermediate layers upstream of the motor-output layer: layer 2/3 (L2/3) and layer 5a (L5a). Although repetitive training often improves motor performance and movement coding by M1 neuronal ensembles, it is unclear how neuronal activities in L2/3 and L5a are reorganized during motor task learning. We conducted two-photon calcium imaging in mouse M1 during 14 training sessions of a self-initiated lever-pull task. In L2/3, the accuracy of neuronal ensemble prediction of lever trajectory remained unchanged globally, with a subset of individual neurons retaining high prediction accuracy throughout the training period. However, in L5a, the ensemble prediction accuracy steadily improved, and one-third of neurons, including subcortical projection neurons, evolved to contribute substantially to ensemble prediction in the late stage of learning. The L2/3 network may represent coordination of signals from other areas throughout learning, whereas L5a may participate in the evolving network representing well-learned movements.

GABA-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion.

γ‐Aminobutyric acid (GABA)‐containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA‐containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67‐immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA‐containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67‐immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67‐ and GABA‐immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA‐containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic. J. Comp. Neurol. 502:113–125, 2007.

The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice

To obtain viable GABAergic interneurons in cerebral cortical slices of adult mice, we investigated the effects of slice cutting solutions on the viability of green fluorescent protein (GFP)-expressing cortical neurons in GAD67-GFP knock-in mice. Almost no nuclei of GFP-positive neurons were labeled with propidium iodide in incubated slices, suggesting that GFP fluorescence was a useful indicator for the viability of GABAergic interneurons. When several cutting solutions were compared with saline-based solution, N-methyl-d-glucamine-based sodium-free solution was most effective to keep the number of GFP-positive neurons near the level of perfusion-fixed brain. GFP-positive neurons in slices cut with sodium-free solution were more numerous in cortical layers V-VI, at 30-60 microm depth from the cut surface and 1-6h after cutting than those with saline-based solution. Furthermore, the number of GFP-positive neurons decreased in the cutting condition of high calcium concentration (5mM) or high temperature (37 degrees C), and GFP fluorescence decreased when cut at 0 degrees C. The present results indicate that cutting the brain at 20 degrees C in sodium-free solution is a method for preparing cortical slices with GABAergic interneurons viable. This method would thus be useful for electrophysiological and morphological studies of cortical interneurons in slice preparations of the adult brain.

Local Connections of Layer 5 GABAergic Interneurons to Corticospinal Neurons

In the local circuit of the cerebral cortex, GABAergic inhibitory interneurons are considered to work in collaboration with excitatory neurons. Although many interneuron subgroups have been described in the cortex, local inhibitory connections of each interneuron subgroup are only partially understood with respect to the functional neuron groups that receive these inhibitory connections. In the present study, we morphologically examined local inhibitory inputs to corticospinal neurons (CSNs) in motor areas using transgenic rats in which GABAergic neurons expressed fluorescent protein Venus. By analysis of biocytin-filled axons obtained with whole-cell recording/staining in cortical slices, we classified fast-spiking (FS) neurons in layer (L) 5 into two types, FS1 and FS2, by their high and low densities of axonal arborization, respectively. We then investigated the connections of FS1, FS2, somatostatin (SOM)-immunopositive, and other (non-FS/non-SOM) interneurons to CSNs that were retrogradely labeled in motor areas. When close appositions between the axon boutons of the intracellularly labeled interneurons and the somata/dendrites of the retrogradely labeled CSNs were examined electron-microscopically, 74% of these appositions made symmetric synaptic contacts. The axon boutons of single FS1 neurons were two- to fourfold more frequent in appositions to the somata/dendrites of CSNs than those of FS2, SOM, and non-FS/non-SOM neurons. Axosomatic appositions were most frequently formed with axon boutons of FS1 and FS2 neurons (approximately 30%) and least frequently formed with those of SOM neurons (7%). In contrast, SOM neurons most extensively sent axon boutons to the apical dendrites of CSNs. These results might suggest that motor outputs are controlled differentially by the subgroups of L5 GABAergic interneurons in cortical motor areas.

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.

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.

Excitatory and inhibitory inputs to PV-positive cortical neurons: A quantitative analysis with BAC transgenic mice

We have developed a novel experimental system for introduction of genetically encoded tools by an adenovirus-mediated gene transfer technique. Here we tested the validity of the system by analyzing the expression pattern of introduced fluorescent proteins. We found that fluorescent cells are pyramidal cells in the cerebral cortex (NeuN-positive, GABA-negative, and GFAP-negative) and Purkinje cells in the cerebellum (IP3R1-positive). Interestingly, the expression pattern in the cortex showed a specific pattern depending on the time when the adenovirus-injection was performed: the injection at embryonic day (E) 12.5 led to the preferential expression in cerebral layer 5/6 neurons (∼90% of expressing neurons), whereas the injection at E14.5 led to the preferential expression in layer 2/3 neurons (∼70% of expressing neurons). Our novel experimental system enables us to introduce genetically encoded tools in specific subtypes of neurons and thus would be a promising way to perform optical recording/manipulation of neural activities in vivo.

Thalamocortical projection neurons in layer VI of the adult rat somatosensory cortex

Brachial plexus (BP) injury is sometimes repaired by nerve crossing for bypassing the injured sites. Successful functional recovery after such operation suggests the presence of some plasticity after nerve crossing. To test this hypothesis, we investigated somatosensory cortical responses after nerve crossing in mice using transcranial flavoprotein fluorescence imaging. Vibratory stimuli applied to the left forepaw elicited bilateral cortical responses. Photo-inactivation of the left cortex suppressed the left and right responses, indicating involvement of callosal fibers for producing the right cortical responses. The right cortical responses after the nerve crossing were reduced in cortex-specific, heterotypic NR1 knockout mice, indicating that experience-dependent plasticity in inter-hemispheric pathways has an important role for functional recovery after nerve crossing in patients with BP injury.