Michiteru Konno

Vesicular glutamate transporter 3-expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei

We previously reported that about 80% of vesicular glutamate transporter 3 (VGLUT3)‐positive cells displayed immunoreactivity for serotonin, but the others were negative in the rat midbrain raphe nuclei, such as the dorsal (DR) and median raphe nuclei (MnR). In the present study, to investigate the precise distribution of VGLUT3‐expressing nonserotonergic neurons in the DR and MnR, we performed double fluorescence in situ hybridization for VGLUT3 and tryptophan hydroxylase 2 (TPH2). According to the distribution of VGLUT3 and TPH2 mRNA signals, we divided the DR into six subregions. In the MnR and the rostral (DRr), ventral (DRV), and caudal (DRc) parts of the DR, VGLUT3 and TPH2 mRNA signals were frequently colocalized (about 80%). In the lateral wings (DRL) and core region of the dorsal part of the DR (DRDC), TPH2‐producing neurons were predominantly distributed, and about 94% of TPH2‐producing neurons were negative for VGLUT3 mRNA. Notably, in the shell region of the dorsal part of the DR (DRDSh), VGLUT3 mRNA signals were abundantly detected, and about 75% of VGLUT3‐expressing neurons were negative for TPH2 mRNA. We then examined the projection of VGLUT3‐expressing nonserotonergic neurons in the DRDSh by anterograde and retrograde labeling after chemical depletion of serotonergic neurons. The projection was observed in various brain regions such as the ventral tegmental area, substantia nigra pars compacta, hypothalamic nuclei, and preoptic area. These results suggest that VGLUT3‐expressing nonserotonergic neurons in the midbrain raphe nuclei are preferentially distributed in the DRDSh and modulate many brain regions with the neurotransmitter glutamate via ascending axons. J. Comp. Neurol. 518:668–686, 2010.

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.

Expression of Gap Junction Protein Connexin36 in Multiple Subtypes of GABAergic Neurons in Adult Rat Somatosensory Cortex

To characterize connexin36 (Cx36)-expressing neurons of the adult rat somatosensory cortex, we examined fluorescence signals for Cx36 messenger RNA (mRNA) in 3 nonoverlapping subpopulations of γ-aminobutyric acid (GABA)ergic interneurons, which showed immunoreactivity for 1) parvalbumin (PV); 2) somatostatin (SOM); and 3) either calretinin (CR), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), or choline acetyltransferase (ChAT). About 80% of PV-, 52% of SOM-, 37% of CR/VIP/CCK/ChAT-immunoreactive cells displayed Cx36 signals across all cortical layers, and inversely 64%, 25%, and 9% of Cx36-expressing neurons were positive for PV, SOM, or CR/VIP/CCK/ChAT, respectively. Notably, although almost all Cx36-expressing neurons in layer (L) 4, L5, and L6 were positive for one of these markers, a substantial proportion of those in L1 (91%) and L2/3 (10%) were negative for the markers tested, suggesting that other types of neurons might express Cx36. We further investigated the colocalization of Cx36 mRNA and α-actinin2 immunoreactivity, as a marker for late-spiking GABAergic neurons, by using mirror-image sections. Surprisingly, more than 77% of α-actinin2-positive cells displayed Cx36 signals in L1-L3, and about 49% and 13% of Cx36-expressing neurons were positive for α-actinin2 in L1 and L2/3, respectively. These findings suggest that all the subtypes of GABAergic interneurons might form gap junctions in the neocortex.

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.

Immunohistochemical analysis of neocortical inhibitory inputs to PV-expressing neurons with BAC transgenic mice

ined the lPAG projection to neurokinin-1 receptor (NK1R) -immunoreactive (-ir) neurons in the ventrolateral medulla, which are distributed predominantly in the pre-Bötzinger complex (pre-BötC) and additionally in the rostral part of the rostral ventral respiratory group (rVRG) region; these NK1R-ir neurons are considered to play an important role for the generation of respiratory rhythm. Using anterograde tracing with biotinylated dextranamine (BDA) and immunohistochemistry for NK1R, we first indicated that NK1Rir neurons in the pre-BötC/rVRG region were embedded in the plexus of axons labeled with BDA injected into the lPAG. When the pre-BötC/rVRG region was examined under an electron microscopy, asymmetrical synapses between these neurons and fibers were observed. Using a combined retrograde tracing with Fluoro-gold (FG) and in situ hybridization technique for vesicular glutamate transporter 2 (VGLUT2) mRNA and glutamic acid decarboxylase 67 (GAD67) mRNA, we secondly demonstrated that vast majority of the lPAG neurons labeled with FG injected into the pre-BötC/rVRG region were positive for VGLUT2 mRNA, but not GAD67 mRNA. Using a combination of anterograde tracing with BDA and immunohistochemistry for VGLUT2 and NK1R, we lastly demonstrated that BDA-labeled lPAG axon terminals showing VGLUT2 immunoreactivity made close apposition with NK1R-ir neurons within the pre-BötC/rVRG region. These data suggest that the lPAG neurons exert excitatory influence upon the NK1R-ir neurons in the pre-BötC/rVRG region for the control of respiratory function.

Efficient visualization of central neurons with lentiviral vectors expressing Red Fluorescent Proteins (RFP)

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.

Dual-Gene Expression in Neurons by Lentivirus with Tet-Off System

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.