Ryohei Tomioka

Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse

γ‐aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase–green fluorescence protein (GAD67‐GFP) knock‐in mouse. The distribution pattern of GFP‐positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67‐negative cells nor any apparent lack of GFP expression in GAD67‐positive neurons in the two GAD67‐GFP knock‐in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock‐in mouse brain. Moreover, we used the knock‐in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP‐positive cells among NeuN‐positive cells (neocortical neurons) was approximately 19.5%. All the CR‐, PV‐, and SS‐positive cells appeared positive for GFP. The CR‐, PV, and SS‐positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR‐, PV‐, and SS‐positive cells among GFP‐positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP‐positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP‐positive cells, many medium‐sized spherical somata emitting intense GFP fluorescence were observed in layer I. J. Comp. Neurol. 467:60–79, 2003.

Differential distribution of vesicular glutamate transporters in the rat cerebellar cortex

The chemical organization of excitatory axon terminals in the rat cerebellar cortex was examined by immunocytochemistry and in situ hybridization histochemistry of vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2). Chemical depletion of the inferior olivary complex neurons by 3-acetylpyridine treatment almost completely removed VGluT2 immunoreactivity from the molecular layer, leaving VGluT1 immunoreactivity apparently intact. On the other hand, neuronal deprivation of the cerebellar cortex by kainic acid injection induced a large loss of VGluT1 immunoreactivity in the molecular layer. In the cerebellar granular layer, both VGluT1 and VGluT2 immunoreactivities were found in mossy fiber terminals, and the two immunoreactivities were mostly colocalized in single-axon terminals. Signals for mRNA encoding VGluT2 were found in the inferior olivary complex, and those for VGluT1 and VGluT2 mRNAs were observed in most brainstem precerebellar nuclei sending mossy fibers, such as the pontine, pontine tegmental reticular, lateral reticular and external cuneate nuclei. These results indicate that climbing and parallel fibers selectively use VGluT2 and VGluT1, respectively, whereas mossy fibers apply both VGluT1 and VGluT2 together to accumulate glutamate into synaptic vesicles. Since climbing-fiber and parallel-fiber terminals are known to make depressing and facilitating synapses, respectively, VGluT1 and VGluT2 might have distinct properties associated with those synaptic characteristics. Thus, it would be the next interesting issue to determine whether mossy-fiber terminals co-expressing VGluT1 and VGluT2 show synaptic facilitation or depression.

Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex

γ‐Aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67‐green fluorescent protein (GFP) knock‐in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP‐positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long‐range projections by tracing GFP‐labeled GABAergic neurons with axon branches traveled rostro‐caudally and medio‐laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.

In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins

A new recombinant virus which labeled the infected neurons in a Golgi stainlike fashion was developed. The virus was based on a replication-defective Sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (pal-GFP). When the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palGFP in the injection site. The labeling was enhanced by immunocytochemical staining with an antibody to green fluorescent protein to show the entire configuration of the dendrites. Thalamocortical axons of the infected neurons were also intensely immunostained in the somatosensory cortex. In contrast to palGFP, when DsRed with the same palmitoylation signal (palDsRed) was introduced into neurons with the Sindbis virus, palDsRed neither visualized the infected neurons in a Golgi stain-like manner nor stained projecting axons in the cerebral cortex. The palDsRed appeared to be aggregated or accumulated in some organelles in the infected neurons. Anterograde labeling with palGFP Sindbis virus was very intense, not only in thalamocortical neurons but also in callosal, striatonigral, and nigrostriatal neurons. Occasionally there were retrogradely labeled neurons that showed Golgi stain-like images. These results indicate that palGFP Sindbis virus can be used as an excellent anterograde tracer in the central nervous system.

Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation

To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1‐ and VGluT2‐immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1‐immunolabeled terminals were smaller than VGluT2‐labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra‐ and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons. J. Comp. Neurol. 465: 234–249, 2003.

Parvalbumin neurons in the forebrain as revealed by parvalbumin-Cre transgenic mice

Neurons expressing the calcium-binding protein parvalbumin (PV) constitute an abundant subpopulation of GABAergic neurons in the cerebral cortex. However, PV is not unique to the GABAergic neurons of the forebrain, but is also expressed in a small number of pyramidal neurons and in a large number of thalamic neurons. In order to summarize the PV neurons in the forebrain, we employed the PV-Cre transgenic mice in the present study. In the progeny of crossbreed between PV-Cre mice and GFP-Cre reporter mice, we found that the GFP-positive neurons include many excitatory neurons in the neocortex and the thalamus as well as GABAergic neurons in the cerebral cortex and basal ganglia. All the reported PV-positive GABAergic neurons in the cerebral cortex and the basal ganglia seemed to be included in the GFP-positive cells. We found GFP-positive layer V pyramidal neurons inhabit a broader neocortical area than was previously reported. They were located in the primary somatosensory, motor, and visual areas. The somatosensory area of the neocortex contained the greatest number of PV-positive pyramidal neurons. A large number of thalamic relay neurons and virtually all the reticular thalamic neurons appeared as GFP-positive. Thalamic relay nucleus and a neocortical area for the same modality corresponded and seemed to contain a characteristic amount of PV-positive excitatory neurons.

Long-Range GABAergic Connections Distributed throughout the Neocortex and their Possible Function

Features and functions of long-range GABAergic projection neurons in the developing cerebral cortex have been reported previously, although until now their significance in the adult cerebral cortex has remained uncertain. The septo-hippocampal circuit is one exception – in this system, long-range mature GABAergic projection neurons have been well analyzed and their contribution to the generation of theta-oscillatory behavior in the hippocampus has been documented. To have a clue to the function of the GABAergic projection neurons in the neocortex, we view how the long-range GABAergic projections are integrated in the cortico-cortical, cortico-fugal, and afferent projections in the cerebral cortex. Then, we consider the possibility that the GABAergic projection neurons are involved in the generation, modification, and/or synchronization of oscillations in mature neocortical neuron activity. When markers that identify the GABAergic projection neurons are examined in anatomical and developmental studies, it is clear that neuronal NO synthetase (nNOS)-immunoreactivity can readily identify GABAergic projection neurons. GABAergic projection neurons account for 0.5% of the neocortical GABAergic neurons. To elucidate the role of the GABAergic projection neurons in the neocortex, it will be necessary to clarify the network constructed by nNOS-positive GABAergic projection neurons and their postsynaptic targets. Thus, our long-range goals will be to label and manipulate (including deleting) the GABAergic projection neurons using genetic tools driven by a nNOS promoter. We recognize that this may be a complex endeavor, as most excitatory neurons in the murine neocortex express nNOS transiently. Nevertheless, additional studies characterizing long-range GABAergic projection neurons will have great value to the overall understanding of mature cortical function.

GABAergic Basal Forebrain Neurons That Express Receptor for Neurokinin B and Send Axons to the Cerebral Cortex

Neurons expressing neurokinin B (NK3) receptor in the basal forebrain region of rats were characterized histochemically by combining immunocytochemistry, in situ hybridization and retrograde labeling, and electrophysiologically by whole‐cell clamp recording. NK3 receptor‐immunoreactive neurons were found in the basal forebrain region including the substantia innominata, where axon terminals immunoreactive for preprotachykinin B, the precursor peptide of neurokinin B (NKB), were densely distributed. More than 90% of NK3 receptor‐expressing neurons in the basal forebrain region showed signals for glutamate decarboxylase mRNA, indicating that almost all NK3 receptor‐expressing neurons were γ‐aminobutyric acid (GABA)ergic neurons. On the other hand, only a few NK3 receptor‐immunoreactive neurons showed immunoreactivity for choline acetyltransferase or parvalbumin in the substantia innominata, ventral pallidum, and globus pallidus, although the distribution of NK3 receptor‐expressing neurons overlapped with those of cholinergic neurons and parvalbumin‐positive neurons. After injection of wheat germ agglutinin into the cerebral cortex, NK3 receptor immunoreactivity was detected in about 25% of retrogradely labeled basal forebrain neurons, indicating that NK3 receptor‐expressing neurons send projection fibers to the cerebral cortex. In the whole‐cell clamp recording study, a selective NK3 receptor agonist evoked membrane depolarization or inward currents with decrease of input impedance in 10 of 100 cortically projecting neurons recorded in the basal forebrain region. Because NKB‐producing striatal neurons send axons selectively to the basal forebrain region (Furuta et al., 2000 ), the present results suggest that the release of NKB by those striatal neurons induces an inhibitory effect on cortical neurons via facilitation of GABAergic basal forebrain neurons expressing NK3 receptor. J. Comp. Neurol. 473:43–58, 2004.

Improved Golgi-like Visualization in Retrogradely Projecting Neurons after EGFP-Adenovirus Infection in Adult Rat and Monkey

An adenovirus vector was generated using a neuron-specific promoter synapsin I and enhanced green fluorescent protein (EGFP) reporter (AdSynEGFP). In addition, two modifications were identified that resulted in robust and reliable retrograde transport and EGFP expression after injection of the virus into three different brain regions in adult rats (medial prefrontal cortex, posterior thalamic nuclear group, and CA1). These are post-injection survival times of 14 days and addition of high concentrations of NaCl (≥600 mM) to the injection buffer. These modifications resulted in obvious improvement in the intensity of the EGFP signal and in the number of labeled cells. Use of anti-EGFP in immunofluorescence or immunoperoxidase processing further enhanced the signal so that Golgi-like filling of dendritic spines and axon collaterals was routinely achieved. Effectiveness of the AdSynEGFP for Golgi-like filling was confirmed in one rhesus monkey with injections in visual area V4. Because of the long-term viability of the infected neurons (at least up to 28 days in rats and 22 days in monkey), this AdSynEGFP is suitable for use in microcircuitry studies in combination with other fluorescently tagged elements, including anterogradely labeled extrinsic projections. The native EGFP signal (without antibody enhancement) may be sufficient for studies involving cultured cells or slices. (J Histochem Cytochem 54:539-548, 2006)

Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions. An immunohistochemical tract-tracing study in the GAD67-GFP knock-in mouse.

It has been reported in the cat and rat that inhibitory premotor neurons, which send their axons to motoneurons of the trigeminal motor nucleus (Vm) are distributed in the reticular regions around the Vm, especially in the supratrigeminal region (Vsup) and the intertrigeminal region (Vint). In the present study, we examined neuronal connections of GABAergic neurons in the Vsup and Vint in the mouse by utilizing the adult heterozygous GAD67-GFP knock-in mouse, in which green fluorescence protein (GFP) is expressed in GABAergic neurons under the control of the endogenous GAD (GAD67) gene promoter [Yanagawa, Y., Kaneko, K., Kanbara, N., Totsuka, M., Yagi, T., Obata, K., 2001. Development of mouse expressing GFP in GABAergic neurons. Neurosci. Res. Suppl. 25, S77; Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J.-I., Obata, K., Kaneko, T., 2003. Green fluorescent protein expression and colocalization with calretinin, parvalbumin and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60-79]. The connections were examined light- and electron-microscopically by combining the anterograde or the retrograde tract-tracing method with the immunohistochemical method for GFP. The data indicated that the Vsup and Vint of the mouse contained GABAergic neurons, which received projection fibers from the marginal layer of the nucleus of the spinal tract of the trigeminal nerve (Vc) on the ipsilateral side and sent their axons to the Vm on the contralateral side. Some of these GABAergic neurons may represent Vm-premotor neurons that receive nociceptive input from the Vc to elicit jaw-opening reflex by inhibiting jaw-closing Vm-motoneurons.