Sex and regional differences in the incorporation of neurons born during song learning in zebra finches

In zebra finches only males sing, and several nuclei controlling song contain more neurons in adult males than in females. The ontogeny of sex differences in neuron number differs across song regions and overlaps with song learning in males. We examined the development of neuron number in several song regions in both sexes. We then determined whether neurons are born and incorporated into song nuclei as sex differences in neuron number emerge, and whether sex or regional differences in the insertion of such neurons may account for differences in the development of these areas. Males add neurons to hyperstriatum ventralis pars caudalis (HVc) and Area X between 20 and 55 d of age. In females there is no change in HVc neuron number during this time, and Area X never appears as a distinct nucleus. In both sexes, 3H-thymidine administration between 20 and 30 d results in neuronal labeling at 55 d in HVc and the region of Area X. However, in these areas the incidence of labeled neurons is higher in males than in females. In contrast to HVc and Area X, sex differences in neuron number in the robustus nucleus of the archistriatum (RA) and the magnocellular nucleus of the neostriatum (MAN) emerge because males retain neurons that are lost in females between 20 and 55 d of age. Accordingly, RA and MAN neurons are not labeled following 3H-thymidine administration between 20 and 30 d of age. These data indicate that sex and regional differences in the ontogeny of song nuclei are related to differences in the incorporation of neurons born during song learning.     

Food rewards modulate the activity of song neurons in Bengalese finches

Vocal learning, a critical component of speech acquisition, is a rare trait in animals. Songbirds are a well‐established animal model in vocal learning research; male birds acquire novel vocal patterns and have a well‐developed ‘song system’ in the brain. Although this system is unique to songbirds, anatomical and physiological studies have reported similarities between the song system and the thalamo‐cortico‐basal ganglia circuit that is conserved among reptiles, birds, and mammals. Here, we focused on the similarity of the neural response between these two systems while animals were engaging in operant tasks. Neurons in the basal ganglia of vertebrates are activated in response to food rewards and reward predictions in behavioral tasks. A striatal nucleus in the avian song system, Area X, is necessary for vocal learning and is considered specialized for singing. We found that the spiking activity of singing‐related Area X neurons was modulated by food rewards and reward signals in an operant task. As previous studies showed that Area X is not critical for general cognitive tasks, the role of Area X in general learning might be limited and vestigial. However, our results provide a new viewpoint to investigate the independence of the vocal learning system from neural systems involved in other cognitive tasks.     

Expression of the potassium‐chloride co‐transporter,KCC2, within the avian song system

Songbirds learn to produce vocalizations early in life by listening to, then copying the songs of conspecific males. The anterior forebrain pathway, homologous to a basal ganglia‐forebrain circuit, is essential for song learning. The projection between the striato‐pallidal structure, Area X, and the medial portion of the dorsolateral thalamic nucleus (DLM) is strongly hyperpolarizing in adults, due to a very negative chloride reversal potential (Person & Perkel, Neuron 46:129–140, 2005). The chloride reversal potential is determined, in part, by the expression level of a neuron‐specific potassium‐chloride cotransporter, KCC2, which is developmentally upregulated in mammals. To determine whether a similar upregulation in KCC2 expression occurs at the Area X to DLM synapse during development, we examined the expression level of KCC2 in adult zebra finches across the song system as well as during development in the Area X – DLM synapse. We demonstrate that KCC2 is expressed in a subset of neurons throughout the song system, including HVC (used as a proper name), robust nucleus of the arcopallium (RA), lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and DLM. The majority of pallidal‐like projection neurons in Area X showed KCC2 immunoreactivity. In adults, KCC2 expression was robust within DLM, and was upregulated between 14 and 24 days post hatching, before the onset of song learning. Light and electron microscopic analysis indicated that KCC2 immunoreactivity is strongly associated with the plasma membrane. Thus, in the song system as in the mammalian brain, KCC2 expression is well placed to modulate the GABA_A reversal potential.     

A novel basal ganglia pathway forms a loop linking a vocal learning circuit with its dopaminergic input

Dopamine has been implicated in mediating contextual modulation of motor behaviors and learning in many species. In songbirds, dopamine may act on the basal ganglia nucleus Area X to influence the neural activity that contributes to vocal learning and contextual changes in song variability. Neurons in midbrain dopamine centers, the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), densely innervate Area X and show singing-related changes in firing rate. In addition, dopamine levels in Area X change during singing. It is unknown, however, how song-related information could reach dopaminergic neurons. Here we report an anatomical pathway that could provide song-related information to the SNc and VTA. By using injections of bidirectionally transported fluorescent tracers in adult male zebra finches, we show that Area X and other song control nuclei do not project directly to the SNc or VTA. Instead, we describe an indirect pathway from Area X to midbrain dopaminergic neurons via a connection in the ventral pallidum (VP). Specifically, Area X projects to the VP via axon collaterals of Area X output neurons that also project to the thalamus. Dual injections revealed that the area of VP receiving input from Area X projects to the SNc and VTA. Furthermore, VP terminals in the SNc and VTA overlap with cells that project back to Area X. A portion of the arcopallium also projects to the SNc and VTA and could carry auditory information. These data demonstrate an anatomical loop through which Area X activity could influence its dopaminergic input.     

Seasonal change in neuron size and spacing but not neuronal recruitment in a basal ganglia nucleus in the avian song control system

Neural plasticity in the song control system of seasonally breeding songbirds accompanies seasonal changes in singing behavior. The volume of Area X, a song control nucleus that forms a portion of the avian basal ganglia, is 75% larger in the spring than it is in the fall. The neuronal basis of the seasonal plasticity in Area X is largely unknown, however. We examined neuronal attributes of Area X in wild adult male song sparrows (Melospiza melodia) captured during the spring and the fall after being implanted for 30 days with osmotic pumps containing [3H]thymidine. We measured the volume of Area X from thionin-stained sections, and neuronal density and number, and average area of the soma from sections labeled with an antibody against Hu, a neuron-specific protein. We sampled two neuron classes: "small" neurons that were most likely striatal-like spiny neurons and "large" neurons, which most likely included pallidal-like projection neurons. We also analyzed seasonal patterns of neuronal recruitment to Area X. The average area of the soma and neuronal spacing for both neuronal classes were greater in breeding birds. There was no difference in total neuron number for both neuronal classes between seasons. The average area of the soma and density and number of newly recruited neurons did not vary across seasons. These results demonstrate that seasonal plasticity in Area X includes changes in neuron size and neuronal density, but not changes in the rate at which new neurons are recruited.     

Thalamostriatal and cerebellothalamic pathways in a songbird,the Bengalese finch

The thalamostriatal system is a major network in the mammalian brain, originating principally from the intralaminar nuclei of thalamus. Its functions remain unclear, but a subset of these projections provides a pathway through which the cerebellum communicates with the basal ganglia. Both the cerebellum and basal ganglia play crucial roles in motor control. Although songbirds have yielded key insights into the neural basis of vocal learning, it is unknown whether a thalamostriatal system exists in the songbird brain. Thalamic nucleus DLM is an important part of the song system, the network of nuclei required for learning and producing song. DLM receives output from song system basal ganglia nucleus Area X and sits within dorsal thalamus, the proposed avian homolog of the mammalian intralaminar nuclei that also receives projections from the cerebellar nuclei. Using a viral vector that specifically labels presynaptic axon segments, we show in Bengalese finches that dorsal thalamus projects to Area X, the basal ganglia nucleus of the song system, and to surrounding medial striatum. To identify the sources of thalamic input to Area X, we map DLM and cerebellar‐recipient dorsal thalamus (DT_CbN). Surprisingly, we find both DLM and dorsal anterior DT_CbN adjacent to DLM project to Area X. In contrast, the ventral medial subregion of DT_CbN projects to medial striatum outside Area X. Our results suggest the basal ganglia in the song system, like the mammalian basal ganglia, integrate feedback from the thalamic region to which they project as well as thalamic regions that receive cerebellar output.     

Neural encoding of auditory temporal context in a songbird basal ganglia nucleus, and its independence of birds' song experience

Some of the most complex auditory neurons known are found in the songbird forebrain, throughout the 'song system', including its basal ganglia nucleus Area X. These cells are selective for the temporal order of the bird's own song (BOS): they typically respond strongly to BOS, but more weakly when the syllable sequence of BOS is played in reverse order (roBOS), indicating that they integrate auditory information over more than single syllables. Here, studying the zebra finch Area X, we found that order selectivity strongly depends on the mean syllable duration of individual songs, decreasing markedly as this duration approaches 150–200 ms. Simply segmenting the same songs differently, creating an increase in average syllable length towards 150 ms, caused a similar decrease in order selectivity. This suggests that song neurons integrate acoustic information over a relatively limited time window, predominantly less than 150 ms. We provided further support for this by showing that a significant fraction of Area X order selectivity was accounted for by the acoustic similarity between each BOS and roBOS, measured using cross-correlation with fixed window sizes, but only when the correlation windows were at least 50 ms and no more than 200 ms long. All the same findings were evident in birds raised without tutor exposure, indicating that tutor learning has little effect on neural mechanisms underlying song temporal selectivity. Our results suggest that song-selective neurons encode much of the temporal context of song using a short, constant time window that is conserved across differences in songs, birds and learning.     

Synaptic connections of thalamo-cerebral vocal nuclei of the canary

The canary vocal nuclei include two systems: hyperstriatum ventrale, pars caudale (HVc), nucleus robustus archistriatalis (RA) and nucleus hypoglossus, pars tracheosyringealis (nXIIts) compose a motor driving system for vocalization. The other group of nuclei including HVc, nucleus X of the lobus parolfactorius (Area X), nucleus dorsointermedius posterior thalami (DIP) and nucleus magnocellularis of anterior neostriatum (MAN) is a modulator of the driving system. The HVc neurons receive mono- or polysynaptic innervation from the MAN and send their fibers to Area X. Axons of Area X terminated in the thalamic nucleus, the DIP, from which neurons extended axons back to the cerebral nucleus, the MAN. Accordingly, the HVc, Area X, DIP and MAN are in a closed loop. Auditory inflow may converge on HVc neurons, partly from either the MAN or Area X during feedback control of the song. Thalamic neurons in the DIP responded to MAN stimulation, and to tonal stimuli, with relatively long latency. The interconnections between the HVc and MAN neurons are presumably central in voco-auditory integration during song-learning.     

Developmental origin and identity of song system neurons born during vocal learning in songbirds

New neurons are added to the forebrain song control regions high vocal center (HVC) and Area X of juvenile songbirds but the identity and site of origin of these cells have not been fully characterized. We used oncoretroviral vectors to genetically label neuronal progenitors in different regions of the zebra finch lateral ventricle. A region corresponding to the mammalian medial and lateral ganglionic eminences generated medium spiny neurons found in Area X and in the striatum surrounding Area X, and at least two classes of interneurons found in HVC. In addition, our experiments indicate that the HVC projection neurons that project into nucleus robust nucleus of the arcopallium (RA) are born locally from the ventricular region immediately dorsal to HVC. The ability to genetically target neuron subpopulations that give rise to different song system cell types provides a tool for specific genetic manipulations of these cell types. In addition, our results suggest striking similarities between neurogenesis in the embryonic mammalian brain and in the brain of the juvenile songbird and provide further evidence for the existence of conserved cell types in the forebrain for birds and mammals.     

An immunohistochemical and pathway tracing study of the striatopallidal organization of area X in the male zebra finch

Area X is a nucleus within songbird basal ganglia that is part of the anterior forebrain song learning circuit. It receives cortical song-related input and projects to the dorsolateral medial nucleus of thalamus (DLM). We carried out single- and double-labeled immunohistochemical and pathway tracing studies in male zebra finch to characterize the cellular organization and circuitry of area X. We found that 5.4% of area X neuronal perikarya are relatively large, possess aspiny dendrites, and are rich in the pallidal neuron/striatal interneuron marker Lys8-Asn9-neurotensin8-13 (LANT6). Many of these perikarya were found to project to the DLM, and their traits suggest that they are pallidal. Area X also contained several neuron types characteristic of the striatum, including interneurons co-containing LANT6 and the striatal interneuron marker parvalbumin (2% of area X neurons), interneurons containing parvalbumin but not LANT6 (4.8%), cholinergic interneurons (1.4%), and neurons containing the striatal spiny projection neuron marker dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) (30%). Area X was rich in substance P (SP)-containing terminals, and many ended on area X neurons projecting to the DLM with the woolly fiber morphology characteristic of striatopallidal terminals. Although SP+ perikarya were not detected in area X, prior studies suggest it is likely that SP-synthesizing neurons are present and the source of the SP+ input to area X neurons projecting to the DLM. Area X was poor in enkephalinergic fibers and perikarya. The present data support the premise that area X contains both striatal and pallidal neurons, with the striatal neurons likely to include SP+ neurons that project to the pallidal neurons.     

Singing-related neural activity in a dorsal forebrain-basal ganglia circuit of adult zebra finches.

The anterior forebrain pathway (AFP) of songbirds, a specialized dorsal forebrain-basal ganglia circuit, is crucial for song learning but has a less clear function in adults. We report here that neurons in two nuclei of the AFP, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and Area X, show marked changes in neurophysiological activity before and during singing in adult zebra finches. The presence of modulation before song output suggests that singing-related AFP activity originates, at least in part, in motor control nuclei. Some neurons in LMAN of awake birds also responded selectively to playback of the bird's own song, but neural activity during singing did not completely depend on auditory feedback in the short term, because neither the level nor the pattern of this activity was strongly affected by deafening. The singing-related activity of neurons in AFP nuclei of songbirds is consistent with a role of the AFP in adult singing or song maintenance, possibly related to the function of this circuit during initial song learning.     

Morphological characterization of HVC projection neurons in the zebra finch (Taeniopygia guttata)

Singing behavior in the adult male zebra finch is dependent upon the activity of a cortical region known as HVC (proper name). The vast majority of HVC projection neurons send primary axons to either the downstream premotor nucleus RA (robust nucleus of the arcopallium, or primary motor cortex) or Area X (basal ganglia), which play important roles in song production or song learning, respectively. In addition to these long‐range outputs, HVC neurons also send local axon collaterals throughout that nucleus. Despite their implications for a range of circuit models, these local processes have never been completely reconstructed. Here, we use in vivo single‐neuron Neurobiotin fills to examine 40 projection neurons across 31 birds with somatic positions distributed across HVC. We show that HVC_(RA) and HVC_(X) neurons have categorically distinct dendritic fields. Additionally, these cell classes send axon collaterals that are either restricted to a small portion of HVC (“local neurons”) or broadly distributed throughout the entire nucleus (“broadcast neurons”). Overall, these processes within HVC offer a structural basis for significant local processing underlying behaviorally relevant population activity.     

Distribution of GABA-like immunoreactivity in the song system of the zebra finch

GABA-like immunoreactivity (GABA-LIR) was mapped in the male and female zebra finch song system using a polyclonal antibody to GABA. GABA-LIR was found throughout the song system in neurons and neuropil of the robust nucleus of the archistriatum (RA), the higher vocal center (HVC), Area X, the magnocellular nucleus of the neostriatum (MAN), and the dorsomedial portion of the nucleus intercollicularis (DM of ICo). Puncta present in the lateral division of MAN (1MAN) may be local interneurons since the only known afferents of 1MAN are from the dorsolateral nucleus of the anterior thalamus (DLM), which did not appear to have any cell bodies with GABA-LIR. Distinct and dense puncta with GABA-LIR were present in DLM, and may be projections from Area X/lobus parolfactorius (LPO). Dramatic sex differences in GABA-LIR distribution were found. Females did not appear to have any GABA-LIR above background in either RA of HVC. Females also did not appear to have a distinct Area X, although they did have many small, lightly staining cell bodies in the corresponding LPO. The distribution of GABA-LIR and sex differences in its distribution suggests that GABAergic neurons may play a role in the acquisition and/or production of song in the zebra finch.     

A comparative study of the behavioral deficits following lesions of various parts of the zebra finch song system: implications for vocal learning

Song production in song birds is controlled by an efferent pathway. Appended to this pathway is a "recursive loop" that is necessary for song acquisition but not for the production of learned song. Since zebra finches learn their song by imitating external models, we speculated that the importance of the recursive loop for learning might derive from its processing of auditory feedback during song acquisition. This hypothesis was tested by comparing the effects on song in birds deafened early in life and birds with early lesions in either of two nuclei--Area X and the lateral magnocellular nucleus of the anterior neostriatum (LMAN). These nuclei are part of the recursive loop. The three treatments affected song development differently, as reflected by various parameters of the adult song of these birds. Whereas LMAN lesions resulted in songs with monotonous repetitions of a single note complex, songs of Area X-lesioned birds consisted of rambling series of unusually long and variable notes. Furthermore, whereas song of LMAN lesioned birds stabilized early, song stability as seen in intact birds was never achieved in Area X-lesioned birds. Early deafness also resulted in poorly structured and unstable song. We conclude that Area X and LMAN contribute differently to song acquisition: the song variability that is typical of vocal development persists following early deafness or lesions of Area X but ends abruptly following removal of LMAN. Apparently, LMAN plays a crucial role in fostering the kinds of circuit plasticity necessary for learning.     

Lucifer Yellow filling of area X-projecting neurons in the high vocal center of female canaries

The avian high vocal center (HVC) is a complex forebrain nucleus that coordinates the sensorimotor integration necessary for song learning and production. It receives auditory and potentially somatosensory input, and sends major projections to vocal motor and anterior forebrain nuclei. The HVC has at least four morphological classes of neurons for which the connectivity remains uncertain. Previous studies have alluded to the functional identity of the cell classes, but none have provided the definitive evidence necessary for subsequent identification of behaviorally relevant changes within known neuronal populations. The cell filling technique we have adapted for use in the song system provides a method by which hodologically identified classes can be described with precision, and song related changes in their morphology can be readily identified. Neurons in female canaries (Serinus canarius) that project to Area X of the anterior forebrain pathway were retrogradely labeled, selectively filled with Lucifer Yellow in a fixed slice preparation, and converted to a Golgi-like stain through an immunocytochemical reaction. We have identified Area X-projecting neurons as belonging to the thick dendrite class of Nixdorf et al. [B.E. Nixdorf, S.S. Davis, T.J. DeVoogd, Morphology of golgi-impregnated neurons in hyperstriatum ventralis, pars caudalis in adult male and female canaries, J. Comp. Neurol. 284 (1989) 337–349] and have shown definitively that they are among the HVC neurons that can receive direct auditory input, as this cell class has short dendrites that extend into the shelf region ventral to HVC that is known to receive auditory inputs. Well-filled axons had collaterals that ramified and terminated within the nucleus, demonstrating a network through which Area X-projecting cells can contribute to intrinsic HVC communication.     

Projections of androgen-accumulating neurons in a nucleus controlling avian song

In zebra finches, androgens stimulate song production and promote growth of the neural regions controlling song. Early exposure to estrogen establishes this sensitivity to androgens and increases the number of androgen-accumulating cells in two song regions, the hyperstriatum ventralis pars caudalis (HVc) and the magnocellular nucleus of the anterior neostriatum. To examine if these regional changes in androgen accumulation could directly influence androgen responsiveness elsewhere in the song system, we combined autoradiographic and retrograde tracing techniques to determine if androgen-accumulating HVc neurons project to other vocal control nuclei. We report here that both major efferent projections from HVc (to Area X and the nucleus robustus of the archistriatum) include a substantial proportion of androgen-accumulating neurons. These data are consistent with the hypothesis that the extent of androgen accumulation in HVc may in turn regulate the androgenic sensitivity of other song regions.     

Long-range GABAergic projection in a circuit essential for vocal learning

The anterior forebrain pathway (AFP) in the passerine song system is essential for song learning but not for song production. Several lines of evidence suggest that area X, a major nucleus in the AFP, forms part of the avian striatum. A key feature of striatal projection neurons is that they use the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Some area X neurons express GABA-like immunoreactivity, but the neurotransmitter phenotype of the projection neurons is largely unknown. To determine whether area X projection neurons are GABAergic, we used immunocytochemistry and confocal microscopy to examine whether these neurons in adult male zebra finches express the GABA synthetic enzyme glutamic acid decarboxylase (GAD). We observed numerous large and small GAD+ somata in area X, and dense GAD+ terminals, but no GAD+ somata in the target of area X, the medial nucleus of the dorsolateral thalamus (DLM). The density of GAD+ terminals in DLM was strongly reduced by ibotenic acid lesions of area X. After tracer injection into the DLM, all of the retrogradely labeled neurons in area X were GAD+. After tracer injection into area X, the vast majority of anterogradely labeled terminals in DLM were GAD+. We conclude that area X neurons projecting to DLM express GAD and are thus likely GABAergic. If this projection is indeed inhibitory, information processing in the AFP is substantially more complicated than previously realized. Moreover, because a GABAergic projection to a thalamic target is reminiscent of pallidal rather than of striatal circuitry, area X may contain both striatal and pallidal components.     

Distribution of vesicular glutamate transporter 2 in auditory and song control brain regions in the adult zebra finch (Taeniopygia guttata)

The songbird brain has a system of interconnected nuclei that are specialized for singing and song learning. Wada et al. (2004; J. Comp. Neurol. 476:44–64) found a unique distribution of the mRNAs for glutamate receptor subunits in the song control brain areas of songbirds. In conjunction with data from electrophysiological studies, these finding indicate a role for the glutamatergic neurons and circuits in the song system. This study examines vesicular glutamate transporter 2 (VGLUT2) mRNA and protein expression in the zebra finch brain, particularly in auditory areas and song nuclei. In situ hybridization assays for VGLUT2 mRNA revealed high levels of expression in the ascending auditory nuclei (magnocellular, angular, and laminar nuclei; dorsal part of the lateral mesencephalic nucleus; ovoidal nucleus), high or moderate levels of expression in the telencephalic auditory areas (cudomedial mesopallium, field L, caudomedial nidopallium), and expression in the song nuclei (HVC, lateral magnocellular nucleus of the anterior nidopallium, robust nucleus of the arcopallium), where levels of expression were greater than in the surrounding brain subdivisions. Area X did not show expression of VGLUT2 mRNA. Nuclei in the descending motor pathway (dorsomedial nucleus of the intercollicular complex, retroambigual nucleus, tracheosyringeal motor nucleus of the hypoglossal nerve) expressed VGLUT2 mRNA. The target nuclei of VGLUT2 mRNA‐expressing nuclei showed immunoreactivity for VGLUT2 as well as hybridization signals for the mRNA of glutamate receptor subunits. The present findings demonstrate the origins and targets of glutamatergic neurons and indicate a central role for glutamatergic circuits in the auditory and song systems in songbirds. J. Comp. Neurol. 522:2129–2151, 2014. © 2013 Wiley Periodicals, Inc.     

Electrophysiological analysis of a songbird basal ganglia circuit essential for vocal plasticity

The discrete, interconnected nuclei of the songbird brain, collectively termed the song system, underlie the learning and production of song. Two main forebrain pathways have been identified that contribute to song production, learning, and adult plasticity. A posterior "motor pathway" including nucleus HVc (used as the proper name), the robust nucleus of the archistriatum (RA) and descending projections to the brainstem, is essential for song production. An "anterior forebrain pathway," arising from HVc, passing through area X of the lobus parolfactorius, the medial portion of the dorsolateral nucleus of the anterior thalamus and the lateral magnocellular nucleus of the anterior neostriatum, and finally terminating in RA, is essential for song learning and adult plasticity. The fact that the lobus parolfactorius is thought to form a part of the avian striatum implies several predictions for the connections of area X and for the properties of its neurons. Here, we review the existing anatomical and electrophysiological data bearing on the nature of area X as a striatal structure. In general, the data strongly favor the notion that area X is striatal. One set of observations, however, is at odds with that idea, and we provide and partially test the hypothesis that area X also contains a pallidal component. We discuss further tests of this idea and implications for thinking of the song system as a basal ganglia loop similar to that described in mammals.