Yasuro Atoji

Afferent and efferent projections of the mesopallium in the pigeon (Columba livia)

The mesopallium is a thick cell plate occupying a substantial portion of the avian dorsal pallium, but its hodology is incompletely known. In pigeons we examined fiber connections of the frontodorsal (MFD) and frontoventral mesopallium (MFV), the ventrolateral mesopallium (MVL), the lateral (MIVl) and medial (MIVm) parts of the intermediate ventral mesopallium, and the caudal mesopallium (MC). MFV, MIVl, and MC connect reciprocally with secondary centers of the trigeminal, tectofugal, and auditory systems, respectively. MVL forms reciprocal connections with both the entopallial core and belt. MFV, MIVl, MVL, and MC receive thalamic inputs different from those of primary sensory pallial regions and have reciprocal connections with the caudolateral nidopallium (NCL) or arcopallium. MIVm has a strong reciprocal connection with the intermediate medial nidopallium. It receives afferents from the visual Wulst, rostral MC, posterior dorsointermediate thalamic nucleus, and caudal part of the posterior dorsolateral thalamic nucleus, is connected reciprocally with the arcopallium, and projects to NCL. MFD has reciprocal connections with the medial frontal nidopallium, arcopallium, posterior pallial amygdala, dorsolateral corticoid area, and projects to the medial part of medial striatum and hypothalamus. These results indicate that six subdivisions of the mesopallium have strong connections with corresponding portions of the nidopallium. In particular, the sensory mesopallial components of MFV, MIVl, MVL, and MC form parallel pathways to the one‐way sensory streams in the nidopallium and make either feedback or feedforward circuits to the secondary centers of the nidopallium. J. Comp. Neurol., 2012.

Afferent and efferent projections of the central caudal nidopallium in the pigeon (Columba livia).

The central caudal nidopallium (NCC) is a large subdivision of the nidopallium in the pigeon brain, but its connectional anatomy is unknown. Here, we examined the connections of NCC by using tract‐tracing methods. Injections of cholera toxin B‐chain (CTB) in NCC labeled many neurons within NCC. Outside NCC, many labeled neurons were found in the dorsal intermediate mesopallium and medialmost part of the medial intermediate nidopallium, with a few in the intermediate (AI) and medial (AM) arcopallium. In the thalamus, labeled neurons were located in the subrotundal nucleus, the shell region of nucleus ovoidalis, and the caudal part of the dorsolateral posterior thalamic nucleus. Injections of biotinylated dextran amine (BDA) in NCC labeled many fibers running rostrocaudally within NCC. Some of these terminated in the dorsal intermediate mesopallium, but the size of the terminal field was smaller than the region of the dorsal intermediate mesopallium that provided the projection to NCC. NCC sent numerous efferents to AI and AM but few to the thalamus. In contrast, after CTB injections in the dorsal intermediate mesopallium, a few neurons were labeled in NCC, but, after BDA injections in the dorsal intermediate mesopallium, large numbers of labeled fibers were seen to project widely throughout NCC. These findings indicate that the flow of information is predominantly from the dorsal intermediate mesopallium to NCC and from there to the arcopallium (AI and AM). The arcopallial outflow to the medial hypothalamus could imply that NCC is involved in neuroendocrine and autonomic functions and is limbic in nature. J. Comp. Neurol. 517:350–370, 2009.

Immunohistochemical localization of vesicular glutamate transporter 2 (vGluT2) in the central nervous system of the pigeon (Columba livia)

Glutamatergic neurons are distributed widely in the telencephalic pallium of birds, but their targets are unknown. In the present study, a polyclonal antibody was produced against pigeon vesicular glutamate transporter 2 (vGluT2) and was used for Western blot and immunohistochemistry to detect projection targets of glutamatergic neurons in the pigeon central nervous system. The molecular weight of vGluT2 was measured at 65 kDa. vGluT2 immunoreactivity was observed in neuropil, but not in neuronal cell bodies or glia. Immunoreactive neuropil generally appeared homogeneous, but fine granules or puncta were found in many areas or nuclei. The telencephalon showed strong immunoreactivity, except for the globus pallidus. In particular, the mesopallium and hippocampal formation revealed the most intense immunostaining. In the diencephalon, vGluT2 immunoreactivity was intense in the dorsal thalamus and hypothalamus. In the midbrain, strong immunostaining was seen in the periaqueductal gray, the dorsal part of the lateral mesencephalic nucleus, and the isthmo‐optic nucleus. The optic tectum showed moderate immunoreactivity. In the cerebellar cortex, glomeruli in the granular layer were intensely immunoreactive, and the molecular layer showed intensely homogeneous immunostaining. In the caudal brainstem, the cochlear magnocellular and angular nuclei showed strongly immunoreactive puncta around neuronal cell bodies. In the spinal cord, the dorsal horn revealed moderate immunoreactivity and the marginal nucleus was strongly immunoreactive. Ultrastructural observations revealed that vGluT2 immunoreactivity is localized in asymmetric, presynaptic terminals. The present results indicate an extensive distribution of glutamatergic projections and circuits in the avian central nervous system. J. Comp. Neurol. 519:2887–2905, 2011.

Expression of the neocortical marker, RORβ, in the entopallium and field L2 of adult chicken

Two opposing hypotheses on the homology of the avian brain suggest that the dorsal ventricular ridge of birds is comparable in certain respects either to the neocortex or to the claustroamygdalar complex of mammals. To help resolve this issue, we examined in adult chicken brains the gene expression of RORβ mRNA, a selective marker for layer IV of mammalian neocortex. RORβ mRNA was expressed in neurons of the chickens visual entopallium and auditory field L2, but not in other regions of the nidopallium, hyperpallium, mesopallium or arcopallium. Together with hodological evidence of direct thalamic projections conveying primary sensory information to the entopallium and field L2, our results support the contention that these two regions are composed of neurons comparable to those in layer IV of mammalian neocortex.

Expression of vesicular glutamate transporter 3 mRNA in the brain and retina of the pigeon.

Vesicular glutamate transporters (vGluTs), which accumulate glutamate into synaptic vesicles, are classified into three subtypes in mammalian brains: vGluT1, vGluT2, and vGluT3. VGluT3 is localized in non-glutamatergic neurons of the brain and retinal amacrine cells. In birds, the vGluT3 genome is found, but its distribution in the brain or retina is unknown. The present study was conducted to analyze vGluT3 cDNA sequence and elucidate its distribution in the pigeon brain and retina. The vGluT3 cDNA comprises 1761bp and showed 95% and 88% identity to the chicken and zebra finch vGluT3 cDNAs, respectively, and 74% identity to human vGluT3 cDNA. In situ hybridization revealed that the vGluT3 mRNA was expressed in neurons of the caudal linear nucleus (LC) of the brain and in amacrine cells of the inner nuclear layer of the retina. A combination of in situ hybridization and serotonin immunohistochemistry revealed three types of stained cells in LC and retina: vGluT3(+)/serotonin(+), vGluT3(+)/serotonin(-), and vGluT3(-)/serotonin(+). The vGluT3(+)/serotonin(+) cells were approximately 22% in LC and 16% in the retina. The present results suggest that the pigeon vGluT3 mRNA is comparable with the mammalian type.

Homology of the mesopallium in the adult chicken identified by gene expression of the neocortical marker cholecystokinin

Studies of gene expression and fiber connections have suggested that the primary visual (entopallium) and auditory (field L2) centers in the avian telencephalon are homologous to layer 4 of extrastriate and auditory neocortices of mammals, respectively. In addition, it has been proposed that the arcopallium contains neurons homologous to layers 5/6 and that the mesopallium may be homologous to superficial neocortical layers, but gene expression evidence for the latter is lacking in adult birds. In the present study using adult chickens we have examined the gene expression of cholecystokinin (CCK) mRNA, a selective marker for layers 2/3 of mammalian neocortex. CCK mRNA was expressed in neurons of the entire mesopallium, but not in any part of the nidopallium. Together with hodological evidence of connections between the mesopallium and the two primary sensory areas, our results are consistent with the suggestion that the mesopallium is comparable to certain superficial layers of mammalian neocortex.

Distribution of vesicular glutamate transporter 2 and glutamate receptor 1 and 2 mRNA in the pigeon retina

Glutamate is an excitatory neurotransmitter in the central and peripheral nervous systems of the vertebrate. The previous studies show the presence of mRNAs of AMPA-type glutamate receptors, GluR1 and GluR2, in the optic tectum of the pigeon, suggesting glutamatergic input from the retina. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) and GluR1 and GluR2 to confirm source of glutamatergic neurons in the pigeon retina by in situ hybridization histochemistry. VGLUT2 mRNA expressed in the inner nuclear layer and ganglion cells, while GluR1 and GluR2 mRNAs were observed in the inner nuclear layer, ganglion cells, and superficial layers of the optic tectum. The results suggest that photoreceptor cells, bipolar cells and ganglion cells are glutamatergic in the avian retina as in mammals.

Distribution of glutamate transporter 1 mRNA in the central nervous system of the pigeon (Columba livia).

Glutamate transporter 1 (GLT1) in glial cells removes glutamate that diffuses from the synaptic cleft into the extracellular space. Previously, we have shown the distribution of glutamatergic neurons in the central nervous system (CNS) of the pigeon. In the present study, we identified cDNA sequence of the pigeon GLT1, and mapped the distribution of the mRNA-expressing cells in CNS to examine whether GLT1 is associated with glutamatergic terminal areas. The cDNA sequence of the pigeon GLT1 consisted of 1889bp nucleotides and the amino acids showed 97% and 87% identity to the chicken and human GLT1, respectively. In situ hybridization autoradiograms revealed GLT1 mRNA expression in glial cells and produced regional differences of GLT1 mRNA distribution in CNS. GLT1 mRNA was expressed preferentially in the pallium than the subpallium. Moderate expression was seen in the hyperpallium, Field L, mesopallium, and hippocampal formation. In the thalamus, moderate expression was found in the ovoidal nucleus, rotundal nucleus, triangular nucleus, and lateral spiriform nucleus, while the dorsal thalamic nuclei were weak. In the brainstem, the isthmic nuclei, optic tectum, vestibular nuclei, and cochlear nuclei expressed moderately, but the cerebellar cortex showed strong expression. Bergmann glial cells expressed GLT1 mRNA very strongly. The results indicate that cDNA sequence of the pigeon GLT1 is comparable with that of the mammalian GLT1, and a large number of GLT1 mRNA-expressing areas correspond with areas where AMPA-type glutamate receptors are located. Avian GLT1 in glial cells probably maintain microenvironment of glutamate concentration around synapses as in mammalian GLT1.

Localization of vesicular glutamate transporter 2 mRNA in the dorsal root ganglion of the pigeon (Columba livia).

Our previous study showed localization of glutamate receptor 1 (GluR1) mRNA in neurons of the pigeon spinal cord, suggesting glutamatergic input from intrinsic and extrinsic origins. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) mRNA to confirm an extrinsic origin of glutamatergic neurons in the dorsal root ganglion (DRG). GluR1 and GluR2 mRNAs were examined in DRG and spinal cord to seek projection regions from VGLUT2 mRNA‐expressing neurons. VGLUT2 mRNA was expressed in most DRG neurons and labelling intensity varied from weakly to intensely. Intense VGLUT2 mRNA expression was mainly seen in medium to large neurons. GluR1 and GluR2 mRNAs were expressed in the dorsal horn and GluR2 mRNA signal was also seen in the marginal nucleus. The results suggest that the pigeon DRG has an extrinsic glutamatergic origin that project to the dorsal horn and marginal nucleus of the spinal cord.

Distribution of Vesicular Glutamate Transporter 2 and Ionotropic Glutamate Receptors in the Auditory Ganglion and Cochlear Nuclei of Pigeons (Columba livia).

Glutamate is a principal excitatory neurotransmitter in the auditory system. Our previous studies revealed localization of glutamate receptor mRNAs in the pigeon cochlear nuclei, suggesting the existence of glutamatergic input from the auditory nerve to the brainstem. This study demonstrated localization of mRNAs for vesicular glutamate transporter 2 (vGluT2) and ionotropic glutamate receptors (AMPA, kainate and NMDA) in the auditory ganglion (AG) and cochlear nuclei (magnocellular, angular and laminar nuclei). VGluT2 mRNA was intensely expressed in AG and intensely or moderately in the cochlear nuclei. The AG and cochlear nuclei showed intense‐to‐moderate mRNA signals for GluA2, GluA3, GluA4, GluK4 and GluN1. These results suggest that the pigeon AG neurons receives glutamatergic input from hair cells and in turn projects to the magnocellular and angular nuclei. Glutamate may play a pivotal role in the excitatory synapse transmission in the peripheral auditory pathway of birds.