The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum)

Abstract

Tyrosine hydroxylase immunohistochemistry is used to demonstrate catecholaminergic neuronal populations in the fore- and midbrain of adult zebrafish (Danio rerio). While no catecholaminergic neurons are found in the midbrain, various immunoreactive populations were found in the diencephalon (hypothalamus, posterior tuberculum, ventral thalamus, pretectum) and telencephalon (preoptic region, subpallium, olfactory bulb). The posterior tubercular catecholaminergic cells include three cytological types (small round, large pear-shaped, and bipolar liquor-contacting cells). Furthermore, the retrograde neuronal tracers DiI or biocytin were applied to demonstrate ascending projections to the basal telencephalon (incl. the striatum). A double-label approach was used — together with tyrosine hydroxylase immunohistochemistry — in order to visualize neurons positive for tyrosine hydroxylase and a retrograde tracer. Double-labeled cells were identified in two locations in the posterior tuberculum (i.e, small round neurons in the periventricular nucleus of the posterior tuberculum and large pear-shaped cells adjacent to it). They are interpreted as the teleostean dopaminergic system ascending to the striatum, since previous work [16] established that no noradrenergic neurons exist in the forebrain of the adult zebrafish.

Introduction

The basal ganglia have recently been convincingly shown to be more uniform among tetrapods regarding their detailed anatomical and neurochemical organization than was previously believed (reviewed in [20]). For example, amphibians do have separate clusters of dopaminergic neurons in the basal midbrain which project to the striatum proper (mesostriatal system) and to the nucleus accumbens (mesolimbic system), respectively [19], [21]. This progress regarding amphibians was due to an improved hodological and neurochemical approach which allowed the delineation of finer forebrain subdivisions (e.g., nucleus accumbens versus striatum proper) and their interrelationship with ascending (e.g., dopamine) systems. The now established fact that neurons composing the ascending dopaminergic system extend from the midbrain into the posterior tubercular area in amphibians [21] makes it promising to look anew at comparable basal ganglia systems in fishes where the situation remains less clear at present for two reasons:

• Anamniote catecholaminergic cells qualifying as candidates for an ascending system to the basal ganglia are not restricted to the basal midbrain, but extend far more anteriorly into the posterior tuberculum.

• Whereas the anamniote lateral subpallium has always been recognized as the seat of the striatum, its finer subdivisions are a matter of debate.

Indeed, in (agnathan) lampreys, catecholaminergic (dopaminergic) cells in the posterior tuberculum were recently reported to project to the area identified much earlier as striatum in these animals [35], [36], [37]. In cartilaginous fishes, dopaminergic cells are abundant in the posterior tuberculum and in the floor of the midbrain; the latter are even referred to as substantia nigra and ventral tegmental area. Unfortunately, no double-labeling study exists in any cartilaginous fish species (see [49] for a review), which would be necessary to demonstrate that such dopaminergic cells project to the lateral subpallium (striatum).

In actinopterygian bony fishes, including teleosts, there are no catecholaminergic cells at all in the basal midbrain [22]. To make matters worse, the location of the lateral subpallium (i.e., striatum) is unclear in teleosts, since their telencephalic hemispheres are everted. Thus, there is considerable historical controversy in the literature on almost every cytoarchitectonic entity identified in the teleostean telencephalon [25], [31], [33], [34], [52]. In contrast, the telencephalic hemispheres of tetrapods and non-actinopterygian gnathostome fishes (e.g., cartilaginous fishes) are evaginated and allow an easier allocation of the basic pallial (e.g., the lateral, i.e., olfactory, pallium and the medial pallium, i.e., the hippocampus) and subpallial divisions (e.g., striatum, septum) (compare [32]).

However, some improvements with regard to recognizing the actinopterygian (teleostean) basal ganglia have been made recently. The dorsal nucleus of area ventralis telencephali (Vd) has been shown to receive direct ascending spinal projections in the zebrafish [2], reminiscent of such a projection to the nucleus accumbens in various mammals [5], [29]. The dorsal (Vd) and ventral (Vv) nuclei of the area ventralis telencephali are further characterized by high concentrations of D₁ dopamine receptor mRNA in the European eel [14] and by many glutamate decarboxylase (GAD) mRNA expressing (i.e., GABA-ergic) neurons in the rainbow trout [1]. These neurochemical features would be expected for striatal structures when compared to the situation in amniotes. Furthermore, Substance P cell bodies/fibers and tyrosine hydroxylase-containing fibers are abundant in the dorsal nucleus of area ventralis of the Senegal bichir [41] and the goldfish [46]. Conversely, dense Substance P fibers and tyrosine hydroxylase-containing cell bodies are present in the posterior tubercular region (posterior tuberal nucleus) in various actinopterygian species. This indicates that reciprocal ascending dopaminergic and descending Substance P-containing projections might exist between the dorsal nucleus of area ventralis telencephali (Vd) and posterior tuberal nucleus, resembling the interrelationship between striatum and substantia nigra in amniotes. Tracer studies are supportive of the existence of such connections [28], [47], [51], [53], since they reveal the teleostean posterior tuberal nucleus — located dorsal to the caudal hypothalamus — to have ascending projections to the telencephalon. Furthermore, catecholaminergic cells were also consistently reported in the posterior tuberculum [22]. However, the existence of such ascending dopaminergic projections has not been investigated and confirmed with a double-label approach in any teleost species so far and must remain hypothetical.

Thus, the goal of the present study is to establish in the zebrafish (Danio rerio), a generally accepted major vertebrate model system:

• the distribution of tyrosine hydroxylase-containing structures in the fore- and midbrain, and

• to visualize with a double-label (immunoreaction and tracing) approach that fraction of tyrosine hydroxylase-containing cells with an ascending basal telencephalic projection.

We report here for the first time teleostean dopaminergic forebrain neurons — located in a rather unexpected dorsal position within the posterior tuberculum — which ascend to the basal telencephalon, and confirm the identification of the dorsal nucleus of area ventralis telencephali (Vd) as the striatum.