immunohistochemical localization of thyrotropin-releasing hormone in the brain of chinook salmon...

THE JOURNAL OF COMPARATIVE NEUROLOGY 345:214-223 (1994)

Immunohistochemical Localization of Thyrotropin-Releasing Hormone

in the Brain of Chinook Salmon (Oncorhynchus tshawytscha)

STUART P. MATZ AND TERRY T. TAKAHASHI Institute of Neuroscience, Eugene, Oregon 97403

ABSTRACT This report describes the distribution of thyrotropin-releasing hormone (TRH) immunore-

activity in the brain of juvenile chinook salmon. TRH-positive cell bodies are observed in the preoptic region of the diencephalon, in the supracommissural nucleus of the ventral telencephalon, and in the internal cellular layer of the olfactory bulb. Immunoreactive fibers occur in the olfactory bulb, the dorsal and ventral telencephalon and were particularly extensive in hypothalamic regions. TRH-positive fibers also are observed in the optic tectum, posterior pituitary and the brainstem.

The cell bodies in the preoptic area reside in the magnocellular preoptic nucleus. The position of these cell bodies along with the location of fibers in the hypothalamus and pituitary is consistent with the role of TRH as a hypothalamic releasing hormone. TRH-positive cell bodies also occur in the supracommissural nucleus of the ventral telencephalon and in the internal cellular layer of the olfactory bulb. The cell bodies in the olfactory bulb may account for some of the fibers in the telencephalon, as there are TRH fibers in the olfactory tracts. The presence of TRH-positive fibers with bouton-like swellings raise the possibility that the TRH peptide may act as a central neurotransmitter of neuromodulator. The results of this study suggest that TRH functions as a modulator of the pituitary activity in salmonids and that TRH is used as a transmitter or modulator in the olfactory system. The presence of TRH-positive somata in the olfactory bulb and ventral telencephalon provides new insights into the comparative anatomy of the salmon telencephalon. c 1994 WiIey-Liss, Inc.

Key words: preoptic area, olfactory pathways, hypothalamic hormones, teleosts

Juvenile chinook salmon (Oncorhynchus tshawytscha), in response to a surge in thyroxine, imprint to the odor bouquet of their homestream. As adults returning from the ocean, they use this imprinted olfactory information to navigate back to their nascent tributary, where they spawn and die (for review, see Hasler and Scholz, 1983). Although this behavior is of obvious importance to the general understanding of neuroplasticity and its regulation by hormones, there is no agreement on how the levels of thyroxine, the triggering hormone, are regulated in salmon, nor is there a detailed description of the organization of the salmon olfactory system.

Thyrotropin-releasing hormone (TRH), a tripeptide amide, was the first hypothalamic releasing factor to be chemically characterized (Boler et al., 1969). In mammals, the involvement of TRH in regulating thyroxine levels is well established (Schally et al., 1969; Yates et al., 1971; Azizi et al., 1974). TRH, produced in the cells of the preoptic area and released into the hypothalamo-hypophysial portal

system, triggers release of thyroid-stimulating hormone (TSH) from the pituitary. TSH, in turn, triggers the release of thyroxine from the thyroid gland. There is some disagree- ment as to whether TRH has a similar function in teleosts (Gorbman and Hyder, 1973; Dickhoff et al., 1978; Eales and Himick, 1988). In fishes, TRH has been implicated in stimulating the release of prolactin (Barry and Grau, 1986), growth hormone (Trudeau et al., 19921, and alpha- melanocyte-stimulating hormone (Lamers et al., 1991) from the pituitary as well as elevating thyroxine levels (Eales and Himick, 1988). These studies suggest that TRH has some role in stimulating the pituitary. However, efforts to localize TRH-positive cell bodies in the preoptic region of fish have been unsuccessful. Hamano et al. (1990) found no immunoreactive TRH cell bodies in the preoptic area of

Accepted January 20, 1994. Address reprint requests to Stuart Matz, Institute of Neuroscience,

Univeristy of Oregon, Eugene, OR 97403.

O 1994 WILEY-LISS, INC.

THYROTROPIN-RELEASING HORMONE IN SALMON

carp, and Batten et al. (1990) were able to localize only a small number of TRH-positive cell bodies in the preoptic area of the sea bass. These results are insufficient to account for the role of TRH in the stimulation of the pituitary.

The present study, therefore, sought to describe the distribution of TRH in the brain of the chinook salmon in an attempt to clarify its role in the stimulation of the pituitary. As we will show, however, TRH immunohistochemistry also proved to be an extremely useful probe with which to analyze the organization of the olfactory system and telencephalon. The distribution of TRH therefore allowed us to address issues regarding the comparative anatomy of the olfactory systems of fishes. A preliminary report of these findings has appeared in abstract form (Matz and Takahashi, 1991).

MATERIALS AND METHODS Animals and tissue preparation

Juvenile chinook salmon, 17-19 months old, were ob- tained from a local hatchery (McKenzie Hatchery, Leaburg, OR). All were from the same stock and were killed during the months of March and April, which is the time when smolt transformation takes place. The “fork length” (measured from the nose to the fork of the tail) of the fish ranged from 14.5 cm to 17.4 cm, and they weighed between 32.5 g and 64.8 g. For all experimental procedures, animals were anesthetized by immersion in 0.1 g/1 MS-222 (3-aminoben- zoic acid ethyl ester, Sigma Chemical Co., St. Louis, MO) and transcardially perfused with cold phosphate-buffered saline (PBS) containing heparin (6.7 mg/100 ml). The perfusion continued with a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were postfixed for 2-4 hours and rinsed in 0.1 M phosphate buffer for 1 hour. Brains were embedded in agar and placed in 30% sucrose until they sank. The brains were sectioned on a cryostat in either the sagittal or transverse plane at 30 km.

The addition of glutaraldehyde to the fixative was crucial for obtaining proper staining. In five specimens in which

ac Dc Dd D1 Dld Dml and

Dm2 DP E ECL Ha ICL OTr PM SAC SFGS SGC SM so SPV v c Vd vl VP vs vv

Abbreviations

anterior commissure central zone of dorsal telencephalon (D) dorsal zone of D lateral zone of D dorsal subdivision of lateral zone of D

medial zones of D posterior zone of D entopeduncular nucleus external cellular layer of olfactory bulb habenula internal cellular layer of olfactory bulb optic tract magnocellular preoptic nucleus stratum album centrale stratum fibrosum et griseum superficiale stratum griseum centrale stratum marginale stratum opticum stratum periventricular commissural nucleus of ventral telencephalon (V) dorsal nucleus of V lateral nucleus of V postcommissural nucleus of V supracommissural of V ventral nucleus ofV

BRAIN 215

glutaraldehyde was not used, there was no staining of either cell bodies or fibers.

Immunohistochemistry A polyclonal antiserum to TRH (Arne1 Products, NY) was

used to localize the TRH immunoreactivity. Sections were incubated overnight at room temperature in the antiserum diluted 1:10,000 in 50 mM PBS containing 0.3% Triton X-100 and blocked with normal goat serum. The antibody was visualized with the AvidinIBiotin kit (Vector Labs, Burlingame, CA) using 3,3’-diaminobenzidine as the chro- mogen. Sections were prepared for light microscopy by dehydration through an alcohol series and cleared with xylene. Alternate sections were used in preincubation controls or stained for Nissl substance with cresyl violet or neutral red.

Hormone preincubation controls Specificity of immunostaining was confirmed by preincu-

bating the antiserum with synthetic TRH (Sigma). TRH is a tripeptide (p-Glu-His-Pro-NH2) with a molecular weight of 362.4 daltons. Preincubation controls with synthetic TRH were carried out at concentrations of 100 FM and’ 890 kM.

Cell measurements Cell bodies in the preoptic region were counted by

examining every other section throughout the hypothalamus under brightfield illumination at 160 x magnification. Only cell bodies with a distinct nucleus were counted. All such cells in alternate sections were counted to give the total number of cell bodies contained at 60 km intervals in each brain. The same considerations were used when counting cell bodies in the ventral telencephalon. In the olfactory bulb, the percentage of TRH-positive cell bodies in the internal cellular layer was determined by counting neurons in sections stained for Nissl substance and compar- ing those numbers with the number of TRH-positive neurons in alternate sections stained for TRH.

RESULTS An overview of the TRH-like immunoreactivity is shown

in Figure 1. Cell bodies were observed only in the preoptic area, ventral telencephalon, and in the olfactory bulb. Immunoreactive fibers were distributed more wideiy.

Location and morphology of TRH-immunoreactive cell bodies

Immunoreactive cell bodies were observed in the preoptic region (Fig. 2). Cell bodies found in rostral positions within the preoptic area tended to be round, whereas those in more caudal positions tended to be oval. In most of the neurons (go%), two processes extend from opposite sides of the soma, giving a bipolar appearance. In the remainder (lo%), multiple processes were visible. As shown in Figure 2, the cells are arranged in a row extending 2,000 km posteriorly from the anterior commissure. The immunoreactive somata were confined to medial positions extending only 400 pm laterally from the ventricular surface (Fig. lb,g-j). This general pattern of staining was consistent in all twenty brains that were examined.

For morphometric purposes, only brains from six fish that were processed under optimal conditions were used. These conditions include adequate exsanguination and

a

C

e

f

i

Figure 1

THYROTROPIN-RELEASING HORMONE IN SALMON

fixation, sectioning without loss of tissue, and optimized immunohistochemical conditions (e.g. antibody and buffer concentrations). The preoptic areas, examined at 60 pm intervals, contained an average of 180 immunoreactive somata per brain (+S S.D.). The diameters of round somata ranged from 10 to 16 pm. For the oval neurons, the length of the short axis ranged from 10 to 14 pm while the length of the long axis ranged from 20 to 24 pm.

In the ventral telencephalon, TRH-immunoreactive (IR) somata were located in the supracommissural nucleus (Figs. lb,g; 3). These were medium-sized neurons, and the immunopositive cell bodies were interspersed among the other cell bodies in the supracommissural nucleus. Most of these neurons appeared round and had single processes that typically radiated in a dorsolateral direction. The diameter of these cell bodies ranged from 10 to 16 prn. There was an average of 87 immunoreactive somata ( + 5 S.D.) within the ventral telencephalon of each brain.

In the olfactory bulb, cells were confined to the internal cellular layer (ICL); no TRH-IR somata were located in the external cellular layer (Figs. lb-d, 4). The TRH-positive cell bodies were dispersed among TRH-negative neurons with the TRH-positive cell bodies constituting approximately 10-15% of the cells in the ICL. These cells were round with diameters ranging from 6 to 12 pm. The axonal processes were not as clearly visible as they were in the preoptic area. Most of the cells were bipolar. Some fibers emanating from these cell bodies were directed toward the glomerular layer of the bulb. Other individual axons could be followed to areas in close proximity to the olfactory tracts. However, our material did not allow for individual axons to be followed into these tracts.

Distribution of TRH-immunoreactive fibers TRH-IR fibers occur in the hypothalamus, the posterior

portion of the pituitary, in the dorsal and ventral regions of the telencephalon, and in the olfactory bulb. Scattered fibers were also observed in the optic tectum and brainstem. Fibers in the olfactory bulb were located in the secondary olfactory fiber (SOF) layer and in the glomerular layer (GL). Fibers in the SOF layer were devoid of terminal swellings, whereas fibers in the GL contained such swellings.

Fibers in the dorsal telencephalon were restricted to two specific regions: the medial zone (Dm) and the posterior zone (Dp). The medial fibers are restricted to the first and second regions of the medial zone (Dml and Dm2) as defined by Northcutt and Braford (1980). The fibers in Dml and Dm2 zone had distinct textures: Dm2 contained coarse fibers that were located dorsally to the fine, more thread- like fibers in Dml. In both areas, terminal-like swellings decorated the length of the fibers (Figs. lb,d-g; 5a). The fibers in the posterior zone of the dorsal telencephalon (Figs. lb,g,h; 5b,c) were also thread-like; however, they were not densely packed, and terminal swellings, although present, were not as prominent as those in Dml and Dm2.

Fig. 1. a: Chart of a parasagittal section identifying the general regions of the brain of a chinook salmon. Planes of section for c-j are indicated. b Parasagittal chart (240 pm lateral) from the brain of a juvenile chinook salmon revealing the distribution of thyrotropin- releasing hormone (TRHI-immunoreactive neurons (dots in the olfactory bulb, ventral telencephalon and preoptic area) and fibers. c-j: Approximate coronal views of TRH immunoreactivity (300 km intervals). TRH-positive cell bodies are seen in sections c,d and g-j.

BRAIN 217

Fig. 2. Thyrotropin-releasing hormone immunoreactivity in the preoptic area. a: Midsagittal section stained for Nissl substance revealing the cell bodies in the preoptic area. Arrowheads indicate row of cells comprising the magnocellular nucleus of the preoptic area. Inset corresponds to Figure lb. b: Adjacent section showing the arrangement of TRH-positive neurons in the preoptic area. These cell bodies are restricted to the magnocellular nucleus of the preoptic area. c: Higher magnification of (b) showing the morphology of TRH-positive neurons in the magnocellular nucleus of the preoptic area. Bars = 200 pm in a,b; 100 pm in c.

Fibers in the ventral telencephalon were difficult to assign to known subdivisions. Ventral telencephalic fibers extended from the caudal olfactory bulb to regions surrounding the anterior commissure, where staining was especially

218 S.P. MATZ AND T.T. TAKAHASHI

Fig. 3. TRH immunoreactivity in the supracommissural nucleus of the ventral telencephalon. a: Coronal section at the level of the anterior commissure showing TRH-positive neurons and fibers in Vs. Inset

corresponds to Figure lg. b: Higher magnification of (a) revealing the morphologV of TRH-positive neurons in Vs. Bars = 200 IJ-m in a; 100 pm in b.

dense. The fibers were not confined to discrete regions as was the case in the dorsal telencephalon, and crossed boundaries of nuclei described by others (Northcutt and Braford, 1980). The staining pattern was more prominent in the ventricular regions: the dorsal nucleus (Vd), ventral nucleus (Vv) and supracommissural nucleus of the ventral telencephalon. Both Vv and Vd contained terminal swellings; however, these swellings were more prominent in Vv. The lateral regions of the ventral telencephalon contained sparse fibers that seemed to radiate out and up toward Dp. These fibers were devoid of terminal swellings.

In the optic tectum, sparsely scattered fibers were located in the stratum opticum (SO), the stratum fibrosum et griseum superficiale (SFGS), the stratum griseum centrale (SGC), and the stratum album centrale (SAC). TRH fibers in the SFGS and SGC are thin and project out radially, whereas the fibers in the SO and SAC form a plexus perpendicular to the radial fibers (Figs. l i j , 6). Terminal swellings, although present, were not prominent in any of these layers.

In the diencephalon, the fiber system extended from the anterior commissure throughout the preoptic area. Fibers also occur in the ventral hypothalamus and branched out laterally in the dorsal and ventral tuberal nuclei. Stained

fibers beaded with swellings were found surrounding many of the TRH-IR neurons in the preoptic region. The diencephalic staining pattern is shown in Figure lb.

Immunoreactive fibers with swellings were localized to the posterior and central regions of the pituitary. Further- more, these fibers were associated with capillary walls adjacent to pituitary cells (Fig. 7). Immunoreactive fibers in the brainstem were found in the medial longitudinal fasciculus and ran the entire length of the brainstem.

Preincubation controls Alternate sections from two animals were used in preincu-

bation controls. Sections processed after the antibody had been preincubated with the TRH antigen show none of the staining described above (Fig. 4b).

DISCUSSION The results of this study suggest that TRH is contained

in the cell bodies of the preoptic area, ventral telencephalon, and olfactory bulb. Neuronal processes containing TRH are found in extensive regions of the olfactory bulb and diencephalon, in discrete regions of the telencephalon, in the pituitary, in the optic tectum, and in the brainstem.

THYROTROPIN-RELEASING HORMONE I N SALMON BRAIN 219

Fig. 4. TRH-positive neurons in the olfactory bulb. a: Coronal section through the olfactory bulb and telencephalon showing TRH- positive somata (arrowheads) in the internal cellular layer of the olfactory bulb. TRH-positive fibers in the telencephalon are also present. Inset corresponds to Figure Id. b Alternate section visualized by the same method when the TRH peptide was preincubated with the

Thyrotropin-releasing hormone as a hypothalamic releasing factor

Two previous studies attempted to localize TRH in the preoptic area of teleosts. In the carp brain, investigators were unable to localize TRH-positive somata in the preoptic area (Hamano et al., 1990). In the sea bass, the investigators located very few TRH-positive somata in the preoptic area, and concluded that these somata could not account for the TRH fibers in the pituitary (Batten et al., 1990). They do not show photographs nor do they specify the subdivision within the preoptic area in which these cell bodies were found. Based on the position and shape of TRH-positive cell bodies in our material, we assign them to the magnocellular preoptic nucleus of Braford and Northcutt (1983). The magnocellular preoptic nucleus contains both parvicellular and magnocellular elements. Parvicellular neurons have diameters of 9-12 pm and occupy ventrorostral positions while the magnocellular neurons have diameters of 20-25 km and occupy dorsocaudal positions. We find TRH- positive somata in both of these regions. Round cell bodies with diameters of 10-16 pm occupy the rostral, parvicellular regions while oval cell bodies with diameters up to 24 pm occupy the caudal, magnocellular regions.

In the salmon brain, the presence of TRH cell bodies in the preoptic area and the presence of fibers in the hypothalamus and pituitary is consistent with a role for TRH as a hypothalamic releasing factor. Furthermore, application of a carbocyanin dye (Dil) to the pituitary in chinook salmon labels magnocellular neurons in the preoptic area (Matz, unpublished data) revealing a connection between these neurons and the pituitary. In mammals, the neural control of thyroxine production is carried out mainly by hypotha-

antibody solution. There is no staining in the olfactory bulb or telencephalon. c: Higher magnification of (a) revealing the morphology of TRH-positive neurons in the internal cellular layer. Arrowheads indicate the same neuron in both (a) and (c). Bars = 200 Fm in a and b; 50 Irm in c.

lamic neurons that produce TRH. TRH is known to stimulate the pituitary to release thyroid-stimulating hormone (TSH) leading to the release of thyroxine from the thyroid gland (Schally et al., 1969; Yates et al., 1971; Azizi et al., 1974). Examining the effects of TRH on TSH release in teleosts, however, is complicated because a reliable assay for TSH is not available. Therefore, any measure of TRH action on the pituitary must be measured indirectly by assaying thyroxine levels after TRH application.

Studies on teleosts are further complicated since teleosts lack a portal system like that of mammals, and the functional relationship between the hypothalamus and the pituitary is not as clearly understood. Moreover, the manner of hypothalamic-pituitary signalling seems to vary among the fishes and there may be multiple pathways within a given species of fish. For instance, gonadotropic releasing hormone (GnRH) neurons in the preoptic area of the black molly (Poecilia Zatipinna) have axons that project into the pituitary where they synapse directly on gonadotropic cells (Peute et al., 1976). Fiber systems in other fish appear to end on the capillary plexus in the posterior portion of the pituitary (Holmes and Ball, 1974). Very little is known regarding TRH signalling in salmon. Ultrastructural evidence from Atlantic salmon suggests that nerve endings termi- nate on the capillary plexus in the posterior region of the pituitary (Fridberg and Ekengren, 1977). Our findings are consistent with the association of nerve terminals and the pituitary vasculature and suggest an anatomical substrate by which TRH can be transported to the pituitary.

Physiological evidence for the role of TRH as a hypothalamic releasing factor in nonmammalian vertebrates has been controversial (Gorbman and Hyder, 1973; Dickhoff et al., 1978; Sawin et al., 1981; Sharp and Klandorf, 1985;


Fig. 5. Extrahypothalamic distribution of TRH-positive fibers. a: Coronal section of the dorsal medial region of the telencephalon (Dm) showing the difference in TRH fiber distribution which corresponds to the nuclear regions Dml and Dm2. There is no staining in the dorsal zone (Dd) of the dorsal telencephalon. Inset corresponds to Figure le.

Jacobs et al., 1988; Licht and Denver, 1988, 1990). It has been reported that synthetic TRH has thyroxine-releasing activity in birds (Sharp and Klandorf, 1985), but evidence in reptiles (Sawin et al., 1981; Licht and Denver, 1988) and amphibians (Jacobs et al., 1988; Licht and Denver, 1990) has been equivocal. In lungfish (Gorbman and Hyder, 1973) and in hagfish (Dickhoff et al., 1978), TRH has been ineffective at inducing thyroxine release. In salmonids, however, TRH has been shown to elevate thyroxine levels (Eales and Himick, 1988). TRH also has been reported to have other actions on the pituitary of teleosts. For instance, TRH has been shown to stimulate the release of prolactin in tilapia (Barry and Grau, 19861, growth hormone in goldfish (Trudeau et al., 1992) and alpha-MSH in tilapia (Lamers et al., 1991). The results of our study and the existence of TRH receptors in the pituitary of teleosts (Burt and Ajah, 1984) are consistent with these findings insofar as they suggest that TRH affects the activity of the pituitary. However, the precise populations of pituitary cells that TRH affects have yet to be identified.

b: Coronal section of TRH fiber distribution in posterior region of the dorsal telencephalon (Dp). Inset corresponds to Figure lh . c: Higher magnification of (b) revealing the morphology of TRH-positive fibers in Dp. Arrowheadsin (b) and (c) indicate the same blood vessel. Bars = 100 p m in a; 200 pm in b; 50 p m in c.

Extrahypothalamic distribution of TRH Our study also demonstrates TRH-positive neurons in

the supracommissural nucleus of the ventral telencephalon (Vs) and in a subset of ICL in the olfactory bulb. This is the first report of TRH-positive neurons in Vs of any teleost. Although the existence of TRH in the olfactory bulb of teleosts has been demonstrated by radioimmunoassay (Jack- son and Reichlin, 19741, there has been no immunocyto- chemical evidence nor has there been data regarding the neuroanatomical locus of the immunoreactivity. We ob- serve TRH immunoreactivity in a subset of neurons in the ICL of the olfactory bulb, suggesting a heterogeneity in the chemical architecture of the olfactory bulb. Whether this heterogeneity extends to the functional level must await electrophysiological analysis.

The differential distribution of telencephalic TRH fibers was useful for studying the organization of the telencephalon. In the dorsal medial (Dm) region of the telencephalon, fibers are distributed into coarse and fine regions. This

THYROTROPIN-RELEASING HORMONE IN SALMON BRAIN 221

Fig. 6. Thyrotropin-releasing hormone immunoreactivity in the optic tectum. Camera lucida drawing of TRH fiber distribution in the optic tectum. Inset corresponds to Figure lj. Bar = 100 pm.

distribution corresponds to the nuclear groups Dml and Dm2 as defined by cytoarchitectonics (Fig. 5a). It is therefore possible to demarcate nuclear groups by the differential distribution of TRH fibers. The origin of TRH fibers in Dm still must be established, but it is possible that they originate from somata in Vs. Horseradish peroxidase (HRP) injections into the Dm region of coastal rockfish reveal retrogradely labelled somata in Vs where we identified TRH-positive cells (Murakami et al., 1983), and lesions of Vs in hime salmon (Oncorhynchus heta) reveal degenerat- ing axons in Dm (Shiga et al., 1985). Results of the lesion study in hime salmon also show that Vs projects to the hypothalamus, preoptic area and most telencephalic regions. Thus, the TRH neurons in vs may also account for some of the TRH fibers in the telencephalon and diencephalon.

The presence of immunoreactive fibers also allows the demarcation of the boundaries of the posterior dorsal telencephalon (Fig. 5b). The origin of these fibers in the posterior dorsal telencephalon (Dp) still must be established, but it is possible that these fibers originate from TRH cell bodies in the ICL of the olfactory bulb. In our material, axon-like processes emanating from these cell bodies are directed toward the olfactory tracts. Further- more, immunoreactive TRH fibers have been reported in the olfactory tract of carp (Hamano et al., 1990) and, in our material, are found in the lateral olfactory tract (LOT). The LOT is thought to carry fibers only from the olfactory bulb (von Bartheld et al., 1984). Although individual axons cannot be followed along their entire length, our material raises the possibility that ICL neurons give rise to TRH- positive fibers in Dp. Projections of the bulb to the dorsal posterior region of the telencephalon have been described in studies that have used the standard axonal tracing methods (Scalia and Ebbesson, 1971; Finger, 1975; Davis et al., 1981; Ebbesson et al., 1981; Levine and Dethier, 1985).

Fig. 7. Thyrotropin-releasing hormone immunoreactivity in the pituitary. a: Camera lucida drawing of TRH fibers along capillaries in the pituitary. b Photomicrograph of TRH-positive fibers in the pituitary. These fibers (arrowheads) appear to be restricted to capillaries adjacent to pituitary cells. Bars = 50 bm.

However, these authors did not determine whether these fibers originate from neurons in the external cellular layer (ECL) or the ICL. We find no TRH neurons in the ECL.

Support for extrinsically projecting ICL neurons comes from studies in catfish and coastal rockfish. In catfish (Bass, 1981a), telencephalic injections of HRP revealed retrogradely labelled neurons in ECL and ICL. In rockfish (Murakami et al., 1983), HRP injections into Dp were found to retrogradely label neurons in both the ECL and ICL. In our material, the TRH neurons in ICL may also project to other olfactory targets in the telencephalon and diencephalon that contain TRH fibers. These areas include the ventral telencephalon, the preoptic area and the hypothalamic regions, all of which are known to receive olfactory projections in teleosts (Bass, 1981a; Levine and Dethier, 1985). However, Vs is also known to project to these regions in salmon (Shiga et al., 1985) and may account for the TRH fibers in these nuclei. The connections of individual nuclei in the salmon brain need to be identified to determine whether TRH fibers in these areas receive their input from olfactory bulb, Vs or preoptic neurons.

The origins of TRH fibers in the optic tectum and brainstem are also unknown at this time. It is unlikely that tectal fibers come from neurons in the olfactory bulb as there is no evidence in teleosts for such a projection.


Similarly, Vs is an unlikely candidate since the only telencephalic region to show input into the tectum in teleosts is the central zone of the dorsal telencephalon (Ito and Kishida, 1977; Bass, 1981b; Murakami et al., 1983). Furthermore, in salmon, HRP injections into Vs do not produce any labelling of fibers in the tectum (Shiga et al., 1985). It may be that these fibers come from TRH neurons in the preoptic area or from some as yet undiscovered TRH neuronal system. The fibers in the medial longitudinal fasciculus of the brainstem are also of unknown origin, but fibers originating in the telencephalon and preoptic area are known to project into this tract.

Comparison of TRH distribution in other vertebrates

The characterization of TRH-positive neurons in distinct nuclei in the salmonid allows us to compare our results with TRH distribution in other vertebrates. Immunohistochemi- cal localization of TRH cell bodies has been carried out in mammals, birds and amphibians, but TRH cell bodies have yet to be localized in the reptilian brain. In the rat brain, TRH cell bodies have been localized in the olfactory bulb, anterior olfactory nucleus, several cortical regions, in the hippocampal formation, in the brainstem and spinal cord as well as the hypothalamus (Hokfelt et al., 1989). In the bird brain, TRH cell bodies were only localized in diencephalic regions including the parvicellular periventricular preoptic area, the lateral hypothalamic area, and the posterior medial hypothalamic nucleus (Jozsa et al., 1988). In the amphibian brain, TRH cell bodies were located in the preoptic area, the dorsal infundibular nucleus, the diagonal band of Broca, and in the medial part of the amygdala (Seki et al., 1983).

Across the vertebrate phylogeny, TRH cell bodies are found in the preoptic area. However, in mammals and in birds, the TRH cell bodies are located in the parvicellular division of the preoptic area while in the salmonid brain they are located in the magnocellular division and in the amphibian brain the exact location of the TRH cell bodies within the preoptic area is left unspecified (Seki et al., 1983). The anatomical and functional significance of this difference is unclear at this time. The magnocellular neurons in the mammalian preoptic area contain oxytocin and vasopressin while the parvicellular neurons contain the hypothalamic releasing factors. Such anatomical distinc- tions have not been made in the salmonid brain. For this reason, it is difficult to correlate the location of TRH neurons with their function. In teleosts it has not been determined that hypothalamic releasing neurons are restricted to the parvicellular neurons. However, the localization of TRH to both magnocellular and parvicellular neurons in salmonids might help explain the multiple releasing activities that TRH appears to have in teleosts.

TRH neurons were also localized in Vs. It is difficult to draw homologies between this nuclei (as well as other telencephalic nuclei) in salmon and nuclei in land vertebrates, due to the evagination of the teleostean brain which results in a mediolateral reversal of the dorsal telencephalon. However, Northcutt and Braford (1980) have proposed that Vs is homologous to the basal amygdala of land vertebrates. The localization of TRH in the amygdala of both rats and amphibians is consistent with this proposal, but in order to further support this homology, the chemical architecture of Vs and its hodology must be examined further.

TRH in the olfactory bulb: Possible anterior olfactory nucleus

The presence of TRH in the ICL of the olfactory bulb and the presence of TRH fibers in the olfactory tracts casts a different light on the structure of the teleostean olfactory bulb. The ICL in teleosts occupies a location similar to that of granule cells in the mammalian bulb, and the two share a number of cytoarchitectural features. This has led to the assumption that they are homologous structures (Satou, 1990). I t is generally believed that these granule cells have no projections to regions outside the bulb. Axonal transport studies in catfish (Bass, 1981a) and rockfish (Murakami et al., 1983) mentioned above, however, indicate that ICL neurons may have external projections. Furthermore, our present study suggests that TRH neurons of the ICL contribute fibers to the lateral olfactory tract. The granule cells of the mammalian bulb and at least some of the cells of the ICL are therefore dissimilar with respect to their connectivities.

Interestingly, Johnston (1911) and Sheldon (1912) had long ago raised the possibility that the ICL not only contains the mammalian equivalent of granule cells, but also contains the equivalent of the anterior olfactory nucleus (AON). In mammals, the AON is a secondary olfactory target which projects to, among other regions, the contralat- era1 olfactory bulb. Sheldon (1912), upon examining the carp brain, concluded that the lateral olfactory tract arises from both mitral cells (the ECL) and the anterior olfactory nucleus (the ICL). Our localization of TRH fibers in the lateral olfactory tract is consistent with this idea. Both Finger (1975) and Bass (1981b) also discussed the possibility of an AON in teleosts. Finger (1975) suggested that the olfactory bulb in teleosts may contain an AON homologue based on his observation that the combined olfactory bulb and AON connections in mammals seem topologically comparable to the olfactory bulb connections in teleosts. Furthermore, he suggested that the placement of the AON within the olfactory bulb could explain the interbulbar connections observed in teleosts but not in mammals. Finger (1975), however, did not describe any extrinsically projecting ICL neurons nor did he pursue this line of inquiry in any more detail.

Bass (1981b) discussed but rejected the placement of the AON within the ICL. He too considered the possibility of an AON due to the existence of the interbulbar connections mentioned by Finger; however, he concluded that this proposition is unsupported since he had shown that mitral cells of the ECL project to the contralateral bulb. We believe that neurons in the ICL may also project extrinsically, possibly to the contralateral bulb, thus appearing similar to neurons in the anterior olfactory nucleus of mammals. We suggest that the extrabulbar-projecting TRH neurons in the ICL of salmon might constitute the AON. The description of TRH-containing neurons in the anterior olfactory nucleus of rats (Hokfelt et al., 1989) supports this idea. However, investigations into the connectivities of the olfactory bulb and o f the TRH neurons in salmon are needed to offer further support.

ACKNOWLEDGMENTS We thank Mark Bennett, Michael Fleming and George

Racette for technical assistance. Gregory Hofeldt provided help with the photographic work. We are also grateful to Richard Francis, Kip Keller, Chuck Kimmel and Carl

THYROTROPIN-RELEASING HORMONE IN SALMON BRAIN 223

Schreck for helpful discussions and for critically reading the manuscript. Also we thank the staff a t McKenzie Hatchery: Dave Rodgers, Loren Jensen, Jim Harvey, Bill Close, Kurt Cummings and Seth Cooney. This work was supported by NIH Systems Physiology Training Program (GM07257).

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