Identification of the origin of catecholaminergic inputs to HVc in canaries by retrograde tract tracing combined with tyrosine hydroxylase immunocytochemistry
Introduction
The nucleus HVc [originally misnamed as Hyperstriatum Ventrale, pars caudale and now sometimes referred to as the high vocal center, see Brenowitz et al. (1997)] is a telencephalic nucleus that appears to be unique to species in the songbird suborder (Nottebohm, 1980, Kroodsma and Konishi, 1991, Ball, 1994, Brenowitz, 1997). It is a key part of a discrete network of interconnected nuclei that are involved in the learning, production and perception of song referred to as the song-control system (Nottebohm, 1980, Nottebohm, 1993, Brenowitz et al., 1997). This neural system includes at least two major circuits. There is a caudal pathway consisting of HVc that projects to the robust nucleus of the archistriatum (RA) of the telencephalon that in turn projects to the intercollicular complex (ICo), in particular the dorsomedial portion of this complex (DM). Both RA and DM project to several medullary components of this circuit including the tracheosyringeal part of the nucleus of the XIIth cranial nerve (nXIIts) that innervates the vocal production organ the syrinx as well as to nucleus retroambigualis (RAm) and the rostral ventral respiratory group of neurons (rVRG) that coordinate respiratory activity with song production (see Fig. 1). This pathway is involved in the motor production of song: lesions to nuclei in the pathway block song production (Nottebohm et al., 1976, Simpson and Vicario, 1990) and both immediate early gene induction studies (Jarvis and Nottebohm, 1997, Kimpo and Doupe, 1997) and electrophysiological studies (Yu and Margolisah, 1996) indicate that neurons in these nuclei are active in association with song production.
A second circuit, the anterior forebrain pathway, has been implicated in song learning, song recognition, sensory feedback of various sorts and the maintenance of crystallized adult song (see Bottjer and Johnson, 1997, Doupe and Solis, 1997, Margoliash, 1997 for reviews). This pathway also includes HVc that projects to area X of the parolfactory lobe (homologue of parts of the mammalian caudate) that in turn projects to the medial part of the dorsolateral thalamic nucleus (DLM). DLM projects to the lateral part of the magnocellular nucleus of the anterior neostriatum (lMAN) that projects to RA (see Fig. 1). The lMAN also projects back to area X.
It has been clear for over 10 years based on a variety of chemical neuroanatomical investigations that HVc and other nuclei in this forebrain circuit must receive inputs that are still undescribed. Studies utilizing high performance liquid chromatography methods (HPLC) carried out on zebra finches (Taeniophygia guttata) have revealed that monoamine neurotransmitters including norepinephrine, epinephrine, dopamine and serotonin are present at high concentrations in many of the forebrain song control nuclei such as HVc (Barclay and Harding, 1988, Sakaguchi and Saito, 1989, Barclay and Harding, 1990). Autoradiographic studies, conducted primarily on European starlings (Sturnus vulgaris), indicate that certain noradrenergic receptors such as the α2 sub-type are present in high densities in HVc and other forebrain song nuclei (Ball, 1994, Bernard and Ball, 1995). High densities of dopamine D1 receptors have been described in area X in starlings (Casto and Ball, 1994). In these cases boundaries of these song control nuclei, can be defined that are consistent with Nissl-defined boundaries, based on the high receptor densities compared to the surrounding brain area. Immunocytochemical studies of tyrosine hydroxylase in zebra finches and canaries (Serinus canaria) have found that the boundaries of HVc can be defined by a higher density of fibers immunoreactive for tyrosine hydroxylase compared to the surrounding neostriatum (Bottjer, 1993, Soha et al., 1996; D. Appeltants et al., unpublished data). Given that a recent study in zebra finches did not identify in HVc a higher density of fibers immunoreactive for the norepinephrine synthezising enzyme, dopamine beta-hydroxylase as compared to the surrounding structures (Mello et al., 1998), it is probable that a significant fraction of the tyrosine hydroxylase-immunoreactive fibers in HVc represent dopaminergic inputs.
All these lines of neuroanatomical evidence indicate strongly that catecholamines and probably indoleamine systems are important actors in the song control system. However, a question remains, where are the cells that synthesize these monoaminergic transmitters that are present in the song control system? Cells synthesizing catecholamines can be identified by localizing the enzyme tyrosine hydroxylase, the rate limiting enzyme for catecholamine synthesis and are readily apparent in the brain of all vertebrate species including birds (Reiner et al., 1994, Smeets and Reiner, 1994). There are ≈17 cell groups that exhibit immunoreactivity for tyrosine hydroxylase (TH) in the avian brain and these are quite similar to what has been observed in other vertebrate species (Reiner et al., 1994). All catecholamines in the brain are synthesized in one of these cell groups. Studies of TH immunoreactivity in the songbird brain provide no evidence (as would be expected) that catecholamines are synthesized in the song control nuclei themselves (Bottjer, 1993, Balthazart and Ball, 1996, Soha et al., 1996; D. Appeltants et al., unpublished data). The exact sources of these catecholaminergic projections to the song system remain largely unknown with the exception of one tract tracing study that has shown that area X receives projections from the ventral tegmental area, a dopaminergic group in mammals and birds (Lewis et al., 1981). Possible connections between HVc in canaries and mid-brain dopaminergic groups have been reported in abstract form (Burd et al., 1986). Norepinephrine is synthesized in a limited range of brain stem nuclei that express TH such as the locus coeruleus and subcoeruleus; these in turn exhibit a complex pattern of forebrain projections. Based on the high levels of norepinephrine that have been measured in the forebrain song control nuclei and the high densities of noradrenergic receptors in nuclei such as HVc and RA one can predict that these nuclei should receive projections from brain stem nuclei such as the locus coeruleus and subcoeruleus.
Identifying these projections is exigent given that a variety of studies have identified a functional significance of these catecholaminergic projections to the song control system. Dave et al. (1998) have found that norepinephrine acting in HVc modulates the auditory responsiveness of neurons in RA. Thus ascending catecholaminergic projections to HVc regulate the sensory responsiveness of cells in RA. Recent studies of the modulation of neural activity in the anterior forebrain pathway as a function of context have implicated ascending catecholaminergic projections in this process as well. Males singing song that is directed at a female exhibit low levels of induction of the immediate early gene ZENK in area X, lMAN and RA while males in isolation exhibit higher levels (Jarvis et al., 1998). Similar findings have been reported for electrophysiological activity in that multi-unit activity is much lower when the birds are engaging in song directed at a female than when they are singing in social isolation (Hessler and Doupe, 1999). Both groups have hypothesized that catecholaminergic projections to these nuclei are responsible for these effects. There is evidence that photoperiod can influence afferent noradrenergic inputs into HVc (Bernard and Ball, 1995) and the manipulation of circulating testosterone levels influences the rates of monoaminergic turnover in the vocal control system (Barclay and Harding, 1988, Barclay and Harding, 1990). No experimental study has to this date identified a specific role for dopamine in the regulation of song behavior or of the activity and morphology of the song system. However, based largely on data from mammalian species, it is clear that dopamine plays a major role in the control of motivation, reinforcement and motor aspects of behavior [see Soha et al. (1996) for additional discussion relevant to songbirds]. Because dopaminergic innervation and dopamine receptors are present within the song control nuclei (see above) and we know that these nuclei are involved specifically in the regulation of different aspects of song behavior, it is expected that dopamine should be involved in the learning, production and/or perception of vocal behavior in songbirds. Seasonal variation in catecholaminergic afferent input into the song control system could also be an important factor regulating seasonal changes in the morphology of the high vocal center.
In order to evaluate the possible function of the catecholaminergic innervation of the song control system, it is first necessary to characterize the neuronal circuitry that links the catecholaminergic system to the vocal control system. Tract-tracing combined with tyrosine hydroxylase immunocytochemistry has been used here to identify the sources of the ascending catecholaminergic projections to the song nucleus HVc in male and female canaries.
Section snippets
Subjects
This study was performed on adult male and female canaries (S. canaria) that were bought from a local dealer in Liège. The birds were photosensitive and maintained in indoor aviaries on a photoperiod of 11L (light):13D (dark) in groups of four to five subjects per cage with food and water available ad libitum. This photoperiodic regimen maintains canaries in a photosensitive state with almost fully developed gonads (Storey and Nicholls, 1976) as was confirmed during the post-mortem inspection
Confirmation and characterization of the injection sites
The pressure injection of a small volume of RLF as described above resulted in discrete injection sites. However, upon inspecting under the microscope the sections from the injected birds it was clear that there were slight variations among birds in the location of the injection as well as the amount of brain surface covered by the tracer. The close apposition of the lateral ventricle to HVc resulted in some diffusion of the tracer into the ventricle that could decrease the total volume of
Discussion
In this study retrograde tract tracing was combined with tyrosine hydroxylase immunocytochemistry to identify in canaries the sources of the ascending catecholaminergic projections to the song control nucleus HVc. The injection site was kept small to insure that it was confined within the boundaries of HVc. These boundaries were defined based both on sections stained for Nissl bodies and on sections stained for TH. All injections that were considered to be within HVc were also associated with
Acknowledgements
This work was supported by grants from the NINDS (NS 35467) to GFB and JB, and by grants from the Belgian FRFC (no. 9.4562.96F), the French Community of Belgium (ARC 99/04-241), and the University of Liège (Crédits spéciaux) to JB. The collaboration between JB and GFB was supported by a NATO grant (CRG973000).
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