Hironobu Sakaguchi

A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes

Songbirds have one of the most accessible neural systems for the study of brain mechanisms of behavior. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene-manipulation tools. To overcome these limitations, we constructed 21 regular, normalized, and subtracted full-length cDNA libraries from brains of zebra finches in 57 developmental and behavioral conditions in an attempt to clone as much of the brain transcriptome as possible. From these libraries, ≈14,000 transcripts were isolated, representing an estimated 4,738 genes. With the cDNAs, we created a hierarchically organized transcriptome database and a large-scale songbird brain cDNA microarray. We used the arrays to reveal a set of 33 genes that are regulated in forebrain vocal nuclei by singing behavior. These genes clustered into four anatomical and six temporal expression patterns. Their functions spanned a large range of cellular and molecular categories, from signal transduction, trafficking, and structural, to synaptically released molecules. With the full-length cDNAs and a lentiviral vector system, we were able to overexpress, in vocal nuclei, proteins of representative singing-regulated genes in the absence of singing. This publicly accessible resource http://songbirdtranscriptome.net can now be used to study molecular neuroethological mechanisms of behavior.

Differential expression of glutamate receptors in avian neural pathways for learned vocalization.

Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds—parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and ‐5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest that neural activity and signal transduction in vocal nuclei of vocal learners will be different relative to the surrounding brain areas. J. Comp. Neurol. 476:44–64, 2004.

The acetylcholine and catecholamine contents in song control nuclei of zebra finch during song ontogeny

The acetylcholine (ACh), dopamine (DA) and noradrenaline (NA) concentrations in the song control nuclei, the magnocellular nucleus of the anterior neostriatum (MAN), the caudal nucleus of the ventral hyperstriatum (HVc) and the robust nucleus of the archistriatum (RA) were measured using high performance liquid chromatography (HPLC) at 5 different ages (30, 40, 50, 60 and 90 days after hatching). ACh concentration of these nuclei increased markedly during the critical period for learning and decreased gradually toward adult age. The change either in the HVc or the MAN preceded that in the RA. The developmental changes of catecholamine concentration of each nucleus were different from those of ACh. DA concentrations in the HVc increased at the same time (between 30 and 40 days after hatching) as ACh increased, whereas concentrations of DA and NA in the other two nuclei did not increase. Combined increment of both ACh and DA occurred only in the HVc and this may contribute to plastic change by dual synaptic modification during the critical period of song learning.

Cholinergic innervation of the song control nuclei by the ventral paleostriatum in the zebra finch: a double-labeling study with retrograde fluorescent tracers and choline acetyltransferase immunohistochemistry

During the sensitive period of song learning, the content of acetylcholine and the enzyme activity of choline acetyltransferase (ChAT) increase remarkably in the song control nuclei of a young male zebra finch. Cholinergic fibers innervate the two main song control nuclei of the forebrain: the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA). The present study combines the retrograde tracer, Fluoro-Red (FRe), with ChAT immunohistochemistry. The results indicate that the cholinergic fibers which innervate the RA and HVC originate from the ventral paleostriatum (VP) in the basal forebrain, and that there is an anterior-posterior topography in the location of the cholinergic neurons in the VP that project to the HVC and RA, although there are a few neurons which project to both nuclei. These findings suggest that the VP is homologous to the nucleus basalis of Meynert of the basal forebrain cholinergic system of mammals which is associated with learning and memory processes, and that the cholinergic neurons in the VP play an important role in avian song learning.

Developmental change of cholinergic activity in the forebrain of the zebra finch during song learning

Song learning in the male zebra finch closely correlates with the development of song-control nuclei, the high vocal center (HVc), the robust nucleus of the archistriatum (RA) and the lateral magnocellular nucleus of the anterior neostriatum (IMAN). We previously found that acetylcholine (ACh) content increases transiently in the RA during the sensitive period of song learning. In the present experiment, development of cholinergic neurons by immunohistochemical methods using monoclonal choline acetyltransferase (ChAT) antibodies, and enzymic activity of ChAT and acetylcholinesterase (AChE) were examined in the RA during the sensitive period of song learning. The developmental change of ChAT activity in the RA was closely parallel to that of the ACh concentration. On the other hand, the density of ChAT-immunoreactive fibers changed little in the RA. This indicates that the transient increase of ACh content during the sensitive period is due to ChAT activity, but not to the increasing density of the cholinergic fibers. The ACh in the RA might be related to plastic changes in the synapse of RA neurons.

Effects of amino acids on cat red nucleus neurons in vitro

SummaryIntracellular records were obtained from neurons in the region of the red nucleus (RN) of cat brain slices. Both EPSPs and IPSPs were recorded in response to local electrical stimulation and these resembled similar electrophysiological responses observed in experiments conducted in vivo. Monosynaptic and polysynaptic IPSPs were observed, suggesting the existence of inhibitory interneurons near or within the RN region.When added to the bathing solution, L-glutamate and L-aspartate depolarized RN neurons with a decrease in input resistance. γ-Aminobutyric acid (GABA) and glycine hyperpolarized the cells with a decrease in input resistance. GABA also elicited a depolarizing response. These amino acid actions had direct postsynaptic effects, since the experiments were conducted in a low Ca2+/high Mg2+ medium which blocked synaptic transmission.

Early song-deprivation affects the expression of protein kinase C in the song control nuclei of the zebra finch during a sensitive period of song learning

MALE zebra finches learn to imitate a tutors song through auditory and motor learning during a sensitive period. The molecular aspects of auditory-dependent learning was investigated using protein kinase C (PKC) as a molecular marker for synaptic plasticity. We found a transient increase in the expression of PKC in the robust nucleus of the archistriatum (RA) during a sensitive period. Furthermore, both early deafening by cochlea removal and song deprivation inhibited the increase of PKC enzyme activity. The results strongly suggest that PKC is an important enzyme related to the synaptic plasticity of RA neurons during a sensitive period of song learning in the zebra finch.

Auditory-vocal cholinergic pathway in zebra finch brain.

: The two main song control nuclei in the zebra finch forebrain, the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA), receive cholinergic innervation from the ventral paleostriatum (VP) of the basal forebrain which may play a key role in song learning. By injecting two kinds of neuroanatomical tracers, we found that a topographically segregated pathway from nucleus ovoidalis (Ov) and nucleus dorsomedialis posterior thalami (DMP) to VP and further to RA and HVC. Ov is known as a major relay in the main ascending auditory pathway. The results suggest that cholinergic neurons in the VP which are responsible for song learning are regulated by auditory information from the Ov.

Sex differences in the developmental changes of GABAergic neurons in zebra finch song control nuclei.

Small neurons in a sexually dimorphic nucleus of the zebra finch, the robust nucleus of the archistriatum (RA), were immunoreactive for γ-aminobutyric acid (GABA). This dimorphism arose from the sex differences in the development of the RA neurons. We examined whether neurons with GABA-like immunoreactivity (GABA-LI) undergo the same developmental changes as the cresyl-violet-stained neurons in the RA. Somata with GABA-LI from male finches became larger during development, while somata with GABA-LI from female finches became smaller, at a rate similar to that in the total population of RA neurons. However, there were marked sex differences in the development of the number of RA neurons with GABA-LI. In the female, neurons with GABA-LI were lost at a rate which was similar to the rate of loss of the total population of RA neurons. On the other hand, in the male, the number of neurons with GABA-LI increased transiently during the sensitive period of song learning, while the total number of RA neurons did not change. This transient increase may be related to the fact that only male birds learn their song during a sensitive period.

Release of endogenous γ-aminobutyric acid from vocalization nucleus, the robust nucleus of the archistriatum of zebra finch in vitro

The release of gamma-aminobutyric acid (GABA) from the robust nucleus of the archistriatum (RA) of the zebra finch was examined in slice preparations. Potassium-induced depolarization caused calcium-dependent release of GABA. GABA release by veratrine was blocked by tetrodotoxin. Electrical stimulation of fibers dorsal to RA also released GABA. There was no sex difference in GABA release per wet weight of RA, while the size of the female RA is smaller than that of the male RA.