Distribution of Thalamic Projection Neurons to the Wulst in the Japanese Quail (Coturnix coturnix japonica)

Distribution of Thalamic Projection Neurons to the Wulst in the Japanese Quail (Coturnix coturnix japonica) Michi YAMADA and Shoei SUGITA Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya-shi 321 (Received April 19, 1993) Abstract The distribution of thalamic projection neurons to the Wulst was examined in the Japanese quail (Coturnix coturnix japonica) using the retrograde transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) method. After injection of WGA-HRP into the unilateral Wulst, retrogradely labeled neurons (RLneurons) were found ipsilaterally in zones A and B of the nucleus dorsolateralis anterior thalami pars lateralis (DLL), the nucleus dorsolateralis anterior thalami pars magnocellularis (DLAmc), the nucleus dorsolateralis anterior thalami pars medialis (DLM) and the nucleus intercalatus thalami (ICT). RL-neurons were also found in the contralateral zone A of the DLL and nucleus tractus septomesencephalicus (nsmt). The ipsilateral DLL had more RL-neurons than those of the contralateral DLL. The thalamo-wulst projection neurons in the ipsilateral DLM and ICT, and the contralateral nsmt have been newly identified in the present study. The distribution of the RLneurons in zones A and B in the DLL, DLAmc and the DLM and ICT closely corresponded to areas which were discovered as retinorecipient zones in our previous reports13,14). Therefore these nuclei could be considered as relay stations in the retino- Wulst pathway. Anim. Sci. Technol. (Jpn.) 64 (10): 978-986, 1993 Key words: Japanese quail, Retrograde transport, Thalamus, WGA-HRP method, Wulst Previous studies s5,8,10,12) have reported that the retinofugal fibers enter the contralateral brain stem and terminate in the nucleus dorsolateralis anterior thalami pars lateralis (DLL), the nucleus dorsolateralis anterior thalami pars magnocellularis (DLAmc), and the nucleus dorsolateralis anterior thalami pars lateralis rostralis (DLA1r). Furthermore, it is well accepted that these dorsolateral anterior thalamic nuclei project to the Wulst4,5,7,8,10-12), which is known as the higher visual center in the avian brain1,7) However, there are differences in the findings among studies of the thalamo-wulst pathway, even used same animal as a pigeon. For example, studies employing retrograde degeneration8) and electrophysiological techniques12) have shown that the nucleus lateralis anterior thalami (LA) serve as the relay station in the retino-wulst pathway. On the other hand, MICELI et al.10), using the retrograde horseradish peroxidase method, reported that there was no neurons projecting to the Wulst in the LA. Therefore, it is important to reexamine the thalamo-wulst relay system in avian. We previously demarcated14) the retinal projections to a group of the dorsal anterior thalamic nuclei and revealed the possibility for thalamo- Anim. Sci. Technol. (Jpn.) 64 (10): 978-986 978 1993

Thalamic Projection Neurons to the Wulst Fig.1. Photographs (a), (b) and (c) show Nissl stained sections of the left dorsal anterior thalamic nuclei. Numbers in each photograph represent the distance from the level of the caudal end of the optic chiasma. Calibration bars are Wulst relay stations to exist in the dorsal anterior thalamic nuclei group of the Japanese quail. The present study was undertaken to clarify the distribution of the thalamo-wulst projection neurons, and the relationship between the retinorecipient area of the dorsal anterior thalamic nuclei and the distribution of the thalamo-wulst projection neurons in the Japanese auail_ Materials and Methods A total of 15 Japanese quail (Coturnix coturnix japonica) of both sexes weighing 80-120g were used in this study. Eleven Japanese quail were used for the retrograde wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) method and the others were used for Nissl preparations. WGA-HRP method : A solution of 2.5% WGA-HRP (Sigma) in physiological saline in phosphate buffer. After a week, the brain which is organized by the hyperstriatum accessorium (HA), hyperstriatum intercalatus superior (HIS) and hyperstriatum dorsale (HD), under sodium pentobarbital anethesia (1.3 mg/30g body weight). After a survival time of two days, all birds were anethetized and 979 perfused intracadially with 200ml of Ringer's solution followed by 500ml of 1% paraformaldehyde and 1.25% glutaraldehyde in a 0.1M phosphate buffer (ph7.4), and finally with 10% sucrose in the same buffer. The brains were stored overnight in phosphate buffer with 20% sucrose solution (ph7.4). Brains were with a freezing microtome and treated with tetramethylbenzidine (TMB)9). Quantitative data were collected from 7 brains. DLL was evenly divided into rostral, middle and caudal parts, and relative distribution of labeled cells in each part had been estimated. In all experiments, the injection sites were checked by drawing with cameralucida in order to confirm that they were restricted to the target nucleus. Nissl preparation : The dorsal part of the brain was exposed by removing the temporal bone and immersed in a 10% formalin solution was completely removed from the skull and reimmersed into the same fixative for a week. The brains were then immersed in a 20% surose phosphate buffer solution for one night, and on a freezing microtome. The sections were

YAMADA and SUGITA Fig.2. Photograph (a) shows the injection site of WGA-HRP into the left Wulst which is organized by HA, HIS and HD. (b) is schematic drawing of injection site (shadow) and subdivision of the Wulst. (c), (d), (e) and (f) show retrogradely WGA-HRP labeled cells in the dorsal anterior thalamic nuclei after WGA-HRP injection. (c), (d) and (f) show labeled cells in the contralateral DLL and nsmt. (e) shows labeled cells in the ipsilateral DLL and DLAmc. Broken line in (e) is showing boundary between zones A and B in DLL. Numbers in each photograph are as in Fig.1. Calibration bars are

Thalamic Projection Neurons to the Wulst Fig. 3. Rostrocaudal series of line drawings of transverse sections through the quail dorsal anterior thalamic nuclei showing distribution of WGA-HRP Wulst (refer to (a) of Fig. 2). Dots are showing RL-neurons. Numbers under the drawings are as in Fig. 1. The calibration bar is 1mm. stained with cresyl violet. The nomenclature of the thalamic nuclei and the hyperstriatum is based on the descriptions in the Japanese quail by EHRLICH and MARK2), HUBER and CROSBY3), and MARTEN and HODOS6). Results After injection of WGA-HRP into the unilateral Wulst (Fig. 2 a, b), retrogradely labeled neurons (RL-neurons) were found ipsilaterally in the lateral portion of the nucleus dorso- 981

YAMADA and SUGITA Fig. 4. Histograms of the relative distribution of WGA-HRP labeled neurons in the ipsilateral and contralateral DLL which were divided into rostral, middle and caudal part. Data is collected from seven brains. A: zone A of DLL B: zone B of DLL C: zone C of DLL AL: ansa lenticularis CA: commissura anterior DLAlr: nucleus dorsolateralis anterior thalami, pars lateralis rostralis DLAmc: nucleus dorsorateralis anterior thalami, pars magnocellularis DLL: nucleus dorsorateralis anterior thalami, pars lateralis DLM: nucleus dorsorateralis anterior thalami, pars medialis GL: nucleus geniculatus lateralis HA: hyperstriatum accessorium HD: hyperstriatum dorsale HIS: hyperstriatum intercalatus superior HV: hyperstriatum ventrale ICT: nucleus intercalatus thalami LA: nucleus lateralis anterior thalami LFB: lateral forebrain bundle nsmt: nucleus tractus septomesencephalicus OM: tractus occipitomesencephalicus OT: tractus opticus QF: tractus quintofrontalis ROT: nucleus rotundus SMT: tractus septomesencephalicus SS: nucleus synencepalicus superficiale VLT: nucleus ventrolateralis thalami V: ventriculus lateralis anterior thalami pars lateralis (DLL), the nucleus dorsolateralis anterior thalami pars magnocellularis (DLAmc), the nucleus dorsolateralis anterior thalami pars medialis (DLM) and the nucleus intercalatus thalami (ICT). RL-neurons were also found in the contralatateral DLL and the nucleus tractus septomesencephalicus (nsmt). lateralis (DLL): In the rostral area, the DLL was located dorsal to the nucleus ventrolateralis thalami (VLT) (Fig. 1 a). More caudally, the DLL comes to lie dorsal to the nucleus rotundus (ROT) and DLAmc (Fig. 1 b). Based on the Nissl preperation, the DLL was divided into the following three zones; A, B and C (Fig. 1 b). Zone A appeared rostral to the ROT, and it was located just dorsal to the ROT in the caudal region. The cell population of zone A was highly dense (Fig. 1 b). Zone B was located dorsomedial to zone A, and had a sparser cell population than that of zone A (Fig. 1 b). Zone C was located dorsomedial to zone B. The density of the cell population of zone C was between that of zone A and zone B (Fig. 1 b). Numerous RL-neurons in the ipsilateral DLL were found in zones A and B (Fig. 2 e, 3 a-d), but no labeled neurons were found in zone C (Fig. 3 d). Altough RL-neurons were distributed throughout the ipisilateral zone A (Fig. 3 a-d), the numbers of these neurons increased toward the caudal level. Especially, large number of RL-neurons distributed in the middle and caudal part of the DLL (Fig. 4). RL-neurons in the caudal part of zone A were found in an area which lies in close contact with the DLAmc and the dorsal part of the ROT (Fig, 2 e, 3 c). Although RL-neurons were found throughout ipsilateral zone B (Fig. 2 e. 3 c, d), zone B had fewer labeled neurons than zone A (Fig. 3 c, d). Considerable number of RL-neurons were found in the rostral part of the contralateral zone A (Fig. 2 c, d, 3 b-d). The numbers of 982

Thalamic Projection Neurons to the Wulst these neurons decreased toward the caudal level of the zone A (Fig. 3 b, c, 4). At the level of the appearance of the ROT, the RL-neurons were found in the dorsal part of zone A (Fig. 3 c). There were no RL-neurons in the contralateral zone B or C (Fig. 3 d). Furthermore, contralaterally RL-neurons were restricted to the rostral DLL in contrast to the wide distribution pattern of the RL-neurons in the ipsilateral DLL (Fig. 3 b-d, 4), magnocellularis (DLAmc): The DLAmc was located medial to the DLL and dorsomedial to the lateral forebrain bundle (LFB) (Fig. 1 b). Although few in number, the RL-neurons were found throughout the ipsilateral DLAmc (Fig. 2 e, 3 b, c). No labeled neurons were found in the contralateral DLAmc. medialis (DLM): The DLM was located medial to the DLL and dorsomedial to the DLAmc and LFB (Fig. 1 b). Retrogradely labeled neurons were sparsely found in the lateral region of the DLM, ipsilaterally (Fig. 3 d). No labeled neurons were found in the contralateral DLM (Fig. 3 c). Nucleus intercalatus thalami (ICT): The ICT was located medial to the ROT, dorsal to the VLT, lateral to the tractus occipitomesencephalicus (OM), ansa lenticularis (AL) and tractus quintofrontalis (QF), and ventral to the LFB (Fig. 1 b). Although few in number RL-neurons were found in the lateral part of the ICT, ipsilaterally, similar to the case for the DLM (Fig. 3 d). No labeled neurons were found in the contralateral ICT (Fig. 3 d). Nucleus tractus septomesencephalicus (ns- MT): Nissl preparations showed that the nsmt was almost entirely comprised of fibers in the rostral part (Fig. 1 a), with neurons increasing in number toward the caudal level (Fig. 1 b). Thus, retrogradely labeled neurons were located in the caudal region of this nucleus contralaterally (Fig. 2 f, 3 d). No labeled neurons were found in the ipsilateral nsnt (Fig. 3 d). Discussion The present study demonstrated that retrogradely labeled neurons (RL-neurons) comprising thalamo-wulst projection system were located in the ipsilateral DLL, DLAmc, DLM and ICT. RL-neurons were also found in the contralateral DLL and nsmt. The distribution area of the RL-neurons in the DLL, DLAmc, DLM and ICT closely corresponded to the retinorecipient areas which were identified in our previous studies13,14) It could be considered that these nuclei could be viewed as relay stations in the retino-wulst pathway. lateralis (DLL): There are a considerable number of studies discussing the thalamo- Wulst projection system in pigeon4,5,8,10-12) and quail15). WATANABE et al.15) showed that the unilateral DLL projected to the bilateral Wulst in the Japanese quail using the anterograde degeneration method. However, they did not show that topographical DLL-Wulst projection system. METER et al.8) and MICELI et al.10) identified the projection from the unilateral DLL to the bilateral Wulst in the pigeon by both retrograde degeneration and horseradish peroxiodase methods. They showed that ventral part of the DLL was strongest thalamo- Wulst projection area. In the present study, RL-neurons were found in the bilateral DLL; i.e., unilateral DLL projected to the bilateral Wulst, with ipsilatelally predominance. On the other hand, it is well known that the DLL is divided into three zones in chick2), pigeon8,10) and quail14). In the present study, RL-neurons were found ipsilaterally in both zones A and B in the DLL, with a greater number of RL-neurons in zone A than Zone B. Zone A in the present study corresponded to the ventral part of DLL in pigeon8). Consequently, present results suggest that the retino-wulst pathways through the DLL in the Japanese quail follow a similar

YAMADA and SUGITA projection system to that of the pigeon. Our previous study14) showed that zone A received stronger input of retionfugal fibers than zone B. Thus, it could be that zone A acts as the main relay station of the retinowulat pathway bilaterally, which suggests that the same visual information is transmitted simultaneously to both hemispheres. Such an organizational structure is consistent with the notion that the retino-dll-wulst pathway plays an important role in the projection of binocular information. Furthermore, since the ipsilateral DLL showed more RL-neurons than the contralateral DLL, it is thought that the ascending connections are bilateral with predominantly ipsilateral bias. magnocellularis (DLAmc): It was reported that unilateral DLAmc neurons projected to the ipsilateral Wulst in the pigeon and those DLAmc-Wulst neurons were a low density10). Although RL-neurons in the present study on the Japanese quail were found throughout the ipsilateral DLAmc, in agreement with MICELI et al.10), the number of RL-neurons was low. Therefore, it could be considered that retino- Wulst pathway via the DLAmc in Japanese quail is the same manner as DLAmc-Wulst projection system in the pigeon. medialis (DLM): Few RL-neurons were found in the ipsilateral lateral part in the DLM. The area where RL-neurons were found in the present study overlapped with retinorecipient areas which were identified in our previous study14), suggesting that the lateral portion of in the DLM act as a relay station in the retino-wulst pathway. However, it is probably a minor nucleus among those included in the thalamo-wulst projection nuclei. Using the HRP method, MICELI et al.10) reported that there were no neurons projecting to the Wulst in the DLM, and failed to find any retinofugal fibers in the pigeon10). Considering these results, it could be suggested that speciesdifferences in the DLM-Wulst projection system. Nucleus intercalatus thalami (ICT): RLneurons were found in the lateral part of the ipsilateral ICT, in nearly the same site identified as the retinorecipient area in our previous study13). Thus, the lateral part of the ICT could be viewed as a relay station of the retino-wulst pathway. Although no studies report the existence of a retinal projection and neurons which project from the ICT to the Wulst in the pigeon and chick, the presence of such a system is reasonable for the following reason: retinofugal fibers terminate in this area13), and neurons projecting to the Wulst lie closely in the same area in the ICT. Nucleus tractus septomesencephalicus (ns- MT): RL-neurons were found in the contralateral nsmt. To the best of our knowledge, no reports exist identifying neurons projecting to the Wulst in this area in pigeon, so this pathway might be specific to the Japanese quail. Indeed, except for a study by HUNT and WEBSTER4), no report has accepted the area as a nucleus. The nsmt can reasonably be regarded as a nucleus because of the presence of considerable numbers of neurons projecting to the Wulst in this area. Other nuclei: MICELI et al.10) reported that the rostrolateral part of the ipsilateral dorsolateral anterior thalamic nucleus (DLAlr) acted as a relay station within the retino-wulst pathway. In the present study, however, no RL-neurons were found in this area. This discrepancy in findings can be attributed to the fact that MICELI et al.10) injected WGA-HRP into the HA, HIS, HD and HV beyond the Wulst, while we restricted the injection site to the Wulst. Actually, when the injection site was accidently deeper than the target depth and distribution of WGA-HRP consequently extended into the HV in the present study, RL-neurons were also found in the DLAlr. MEIER et al.8), using retrogradely degeneration, and MIHAILOVIC et al.12), using electro- 984

Thalamic Projection Neurons to the Wulst physiological techniques, reported that the lateral anterior thalamic nucleus (LA) was the relay station of the retion-wuist pathway in the pigeon. MICELI at al.10) reported that there were no reports projecting to the Wulst in the LA by the HRP method. Consequently, it could be considered that the LA is not a relay station of the retino-wulst pathway, in accordance with neuroanatomical studies. However, the LA receives strong retinofugal input, suggesting the possibility that the LA projects to the Wulst via other brain stem nuclei. In the present study, projection system from the thalamic nuclei to each subdivision of the Wulst coud not clear since it was hard to make a restricted injection site in each subnucleus of the Wulst. Referances 1) DENTON, C. J., Topography of the hyperstriatal visual projection area in the young domestic chicken. Exp. Neurol., 74: 482-498. 1981. 2) EHRLICH, D. and R. MARK, An atlas of the primary visual projections in the brain of the chick, Gallus gallus. J. Comp. Neurol., 223: 592-610. 1984. 3) HUBER, G.C. and E.C. CROSBY, The nuclei and fiber pathways of the avian diencephalon, with consideration of telencephalic and center mesencephalic centers connections. J. Comp. Neurol., 48: 1-225. 1929. 4) HUNT, S.P. and K.E. WEBSTER, Talamohyperstriate interrelations in the pigeon. Brain Res., 44: 647-651. 1972. 5) KARTEN, H.J., W. HODOS, W.J.-H., NAUTA and A.M. REVZIN, Neuronal connections of the "Visual Wulst" of the avian telencephalon. Experimental studies in the pigeon (Columba livia) and owl (Speotyto cunnicularia). J. Comp. Neurol., 150: 253-278. 1973. 6) KARTEN, H.J., and W. HoDOS, A stereotaxic atlas of the brain of the pigeon (Columba livia). Baltimore, John Hopkins Press University Press. 1967. 7) KUENZEL, W.J., Neuroanatomical substrates involved in the control of food intake. Poultry Sci., 68 : 926-937. 1989. 8) MEIER, R.E., J. MIHAILOVIC and M. CUENOD, Thalamic organization of the retino-thalamo-hyperstriatal pathway in the pigeon (Columba livia). Exp. Brain Res., 19: 351-364. 1974. 9) MESULAM, M.M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry : a non-carcinogenic blue reaction-product with supperior sensitibity for visualizing neuronal afferents and efferent. J. Histochem. Cytochem., 26: 106-117. 1978. 10) MICELI, D.J. PEYRICHOux and J. REPERANT, The retino-thalamo-hyperstriatal pathway in the pigeon (Columba livia). Brin Res., 100: 125-131. 1975. 11) MICELI, D. and J. REPERANT, Thal.amo-- hyperstriatal projections in the pigeon (Columba livia) as demonstrated by retrograde double-labeling with fluorescent tracers. Brain Res., 245 : 365-371. 1982. 12) MIHAILOVIC, T.M.,R. PERISIC, R. BERGONZL and R.E. MEIER, The dorsolateral thalamus as a relay in the retino-wulat pathway in pigeon (Columba livia). An elertrophysiological study. Exp. Brain Res., 21 : 229-240. 1974. 13) YAMADA, M. and. S. SUGITA, Retinal projection into the hypothalamus and ventral anterior thalamus in the Japanese quail (Coturnix coturnix japonica). Anim. Sci. Technol. (Jpn.), 64: 45-53. 1993. 14) YAMADA, M. and S. SUGITA, Retinal projection to the dorsal anterior thalamic area in the Japanese quail (Coturnix coturnix japonica). Anim, Sci Technol. (Jpn.), 64 : 608-613. 1993. 15) WATANABE, M., H. ITO and H. MASAI, Cytoarchitecture and visual receptive neurons in Wulst of the Japanese Quail (Coturnix coturnix japonica). J. Comp. Neurol., 213: 188-198. 1983. 985

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