MICROSCOPY RESEARCH AND TECHNIQUE 52:520–533 (2001)

Neural Input and Neural Control of the Subcommissural Organ

1 2 1 ANTONIO J. JIME´ NEZ, * PEDRO FERNA´ NDEZ-LLEBREZ, AND JOSE MANUEL PE´ REZ-FI´GARES 1Departamento de Biologı´a Celular y Gene´tica, Facultad de Ciencias, Universidad de Ma´laga, Ma´laga, Spain 2Departamento de Biologı´a Animal, Facultad de Ciencias, Universidad de Ma´laga, Ma´laga, Spain

KEY WORDS serotonin; GABA; monoamines; pineal organ ABSTRACT The neural control of the subcommissural organ (SCO) has been partially character- ized. The best known input is an important serotonergic innervation in the SCO of several mammals. In the rat, this innervation comes from raphe nuclei and appears to exert an inhibitory effect on the SCO activity. A GABAergic innervation has also been shown in the SCO of the rat and frog Rana perezi. In the rat, GABA and the enzyme glutamate decarboxylase are involved in the SCO innervation. GABA is taken up by some secretory ependymocytes and nerve terminals, coexisting with serotonin in a population of synaptic terminals. Dopamine, noradrenaline, and different neuropeptides such as LH- RH, vasopressin, vasotocin, oxytocin, mesotocin, substance P, ␣-neoendorphin, and galanin are also involved in SCO innervation. In the bovine SCO, an important number of fibers containing tyrosine hydroxylase are present, indicating that in this species dopamine and/or noradrenaline-containing fibers are an important neural input. In Rana perezi, a GABAergic innervation of pineal origin could explain the influence of light on the SCO secretory activity in frogs. A general conclusion is that the SCO cells receive neural inputs from different neurotransmitter systems. In addition, the possibility that neurotransmitters and neuropeptides present in the cerebrospinal fluid may also affect the SCO activity, is discussed. Microsc. Res. Tech. 52:520–533, 2001. © 2001 Wiley-Liss, Inc.

INTRODUCTION 5,6-dyhydroxytriptamine or 5,7-dyhydroxytriptamine The subcommissural organ (SCO) is an ependymal (5,6-DHT; 5,7-DHT; Møllgard et al., 1978; Møllgard structure that covers and penetrates the posterior com- and Wiklund, 1979) have convincingly demonstrated missure (Rodrı´guez et al., 1987, 1992, 1998). The SCO the innervation of the SCO by serotonergic fibers. cells are arranged into two layers, ependyma and hy- In the rat SCO, serotonin is located in well-differen- pendyma. tiated axoglandular synapses with both ependymocytes The secretory activity of this brain gland, as other and hypendymocytes (Bouchaud and Arluison, 1977; body glands, is expected to be controlled by means of Bouchaud, 1979, 1993; Møllgard et al., 1978; Møllgard molecular signals. Two general ways of control for a and Wiklund, 1979). Synaptic contacts are on the basal gland have been classically considered: the humoral processes and laterobasal portions of the ependymal and the neural inputs. A confirmed way of control of the secretory cells. Bouchaud (1979), using high-resolution SCO is by synaptic contacts using neurotransmitters or radioautography in the rat, concluded that the seroto- neuropeptides. Although the SCO has a rich vascular- nergic terminals represent approximately 75% of the ization, it presents a blood-brain barrier that restricts synaptic contacts. Therefore, the remaining 25% of the access of blood factors (Bouchaud, 1975; Krisch, non-labeled synaptic contacts contain another neuro- 1993; Porier et al., 1983; Rodrı´guez et al., 1998). This transmitter. Mo¨llgard and Wiklund (1979), Wiklund gland presents another humoral domain with the in- and Møllgard (1979), Bouchaud (1979; 1993) and ternal (ventricular) and external (subarachnoidal) ce- Bouchaud and Bosler (1986) agree with respect to the rebrospinal fluid (CSF) compartments. Thus, the pos- ultrastructural characteristics of the serotonergic syn- sibility that the SCO receives signals from the CSF has aptic contacts in the rat SCO. Synaptic contacts dis- to be considered. played clear vesicles, 40–55 nm in diameter, frequently pleomorphic and less than 1% of large granular vesi- SEROTONERGIC INNERVATION cles. On the other hand, the non-serotonergic terminals OF THE SUBCOMMISSURAL ORGAN contained large granular vesicles and clear vesicles The first evidence of a serotonergic innervation of the with an average diameter of 50 nm, occasionally dis- SCO comes from histochemical studies carried out in playing a paracrystaline arrangement (Bouchaud, rats using formaldehyde-induced fluorescence that 1979, 1993; Bouchaud and Bosler, 1986). The postsyn- showed a dense serotonergic plexus (Bjo¨rklund et al., aptic ependymal cytoplasm showed electron-dense ma- terial attached to the postsynaptic membrane, confer- 1972; Bouchaud and Arluison, 1977; Fuxe, 1965; Møll- Ј gard and Wiklund, 1979; Wiklund, 1974). Such a mas- ring to it the aspect of an asymmetrical Gray s Type-I sive innervation was later confirmed by means of im- munocytochemical studies (Matsuura et al., 1989; Ueda et al., 1988). Ultrastructural radioautography Contract grant sponsor: DGICYT; Contract grant number: PB96-0696; Con- using 3H-serotonin (Bouchaud and Arluison, 1977; tract grant sponsor: FIS; Contract grant number: 98/1508. Bouchaud, 1979) and the ultrastructural demonstra- *Correspondence to: Antonio J. Jime´nez. Departamento de Biologı´a Celular y Gene´tica, Facultad de Ciencias, Universidad de Ma´laga, Ma´laga, Spain, tion of degeneration of nerve terminals after intraven- E-29071. E-mail: [email protected] tricular administration of neurotoxic drugs such as Received 3 March 2000; Accepted 26 May 2000

© 2001 WILEY-LISS, INC. INNERVATION OF THE SCO 521 synapse (Bouchaud and Bosler, 1986; Bouchaud, 1993; comes from the nuclei raphe centralis superior and Wiklund and Møllgard, 1979). raphe dorsalis, each nucleus contributing about one- third of the input (Le´ger et al., 1983). These experi- Comparative Studies of the Serotonergic ments also describe that the nucleus raphe centralis Innervation of the Subcommissural Organ superior contributes with thin fibers that innervate the The studies of the serotonergic innervation in differ- nuclear level of the ependymal layer of the SCO, ent mammalian species have resultes in contradictory whereas the nucleus raphe dorsalis contributes with results. Matsuura et al. (1989) distinguished three thicker fibers innervating the hypendymal cells. Later, morphological types of serotonergic innervation. Ac- a study using the neuronal tracer Phaseolus vulgaris- cording to these authors, type I is observed in the rat leucoagglutinin (PHA-L) applied in different raphe nu- and the Japanese squirrel, it is characterized by a clei confirmed the origin from the nucleus raphe dor- dense serotonergic plexus located at the basal portion salis (Mikkelsen et al., 1997). The innervation of the of the SCO ependymal cells (Fig. 1). In type II, the rat SCO by serotonergic axon terminals has also been innervation is distributed among the cells of the SCO partially prevented by X ray-irradiation on the brain- ependyma and can be found in the dog and cat. In the stem during the early postnatal period (Delhaye- dog, this innervation is more pronounced in the rostral Bouchaud and Bouchaud, 1993). portion of the SCO, and terminals resembling those According to Marcinkiewicz and Bouchaud (1986) described in the rat can be ultrastructurally observed and Bouchaud and Bosler (1986), the rat SCO is (Matsuura and Sano, 1987). Type III corresponds to the reached by the serotonergic innervation in a rostrocau- absence of such an innervation, and it has been de- dal direction from the lamina intercalaris. Mikkelsen scribed in the monkey, guinea pig, and adult mouse. et al. (1997) have reported, using PHA-L in the rat, Wiklund et al. (1977) have also observed that the SCO that the ascending projections from nucleus raphe dor- of the Mongolian gerbil and rabbit lacks of a serotoner- salis extended mainly within or along the ependyma of gic innervation. In 20-day-old mice of the C57BL/10J the dorsal part of the aqueduct directly toward the SCO strain we have found, using immunocytochemistry region. Finally, labeled fibers reach the SCO from the against serotonin at the light-microscopic level, the ependyma in its lateral portions and from the posterior existence of a dense plexus of labeled fibers in close commissure. contact with the SCO, displaying a similar pattern to the serotonergic innervation of the rat (Figs. 2–4). We Deafferentation and Reinnervation of the have also detected an important number of immunore- Subcommissural Organ active fibers contacting the basal portion of the bovine The effect of the serotonergic innervation has been secretory SCO ependymocytes (Fig. 5). Some of the studied in the rat by means of selective destruction of labeled fibers penetrated into the ependymal layer serotonergic fibers using neurotoxic indolamines such near the apical border. as 5,6-DHT (5,6-dihydroxytryptamine) or 5,7-DHT In non-mammal species such as the frog Rana perezi (5,7-dihydroxytryptamine). Mo¨llgard and Wiklund (Jime´nez et al., 2000) or the goldfish Carassius auratus (1979) carried out such a destruction and found that (Pe´rez-Fı´gares et al., 1993, Jime´nez et al., 1993) a after the first 3 days, ultrastructural changes were serotonergic innervation has not been detected. present in synaptic profiles innervating the SCO. Møll- gard and collaborators (Møllgard et al., 1978; Møllgard Ontogenetic Development of the and Wiklund, 1979) described a sequence of changes in Serotonergic Innervation the rat SCO ultrastructure that could be related to Different ontogenetic studies carried out in the rat, increased synthetic and secretory activities. According gerbil, mouse, rabbit, and cat agree with the fact that to these authors, such changes consisted in an hyper- neonatal specimens lack a serotonergic innervation tropy of the endoplasmic reticulum, a great increase in (Marcinkiewicz and Bouchaud, 1986; Matsuura et al., the number of secretory granules and increased release 1989; Wiklund, 1974; Wiklund et al., 1977). In con- of secretory material into the CSF. These changes were trast, the neonatal SCO of the dog (Matsuura and apparently still detected after 8 months. Although a Sano, 1987) and of the semi-desertic rodent Meriones reinnervation of the SCO is observed after one month, shawi (Laalaoui et al., 1996) is already innervated by this is non-monoaminergic and the contacts newly serotonergic fibers that complete their development at formed differ ultrastructurally from the serotonergic 3 months and 2 weeks of age, respectively (Matsuura synapses formerly described (Wiklund and Møllgard, and Sano, 1987). In the rat, serotonergic fibers ap- 1979). peared to establish synaptic contacts with the rostral A study about the reinnervation of serotonin fibers in portion of the SCO on the third postnatal day the denervated SCO in rats using fetal raphe trans- (Bouchaud and Bosler, 1986; Marcinkiewicz and plants has shown a serotonergic reinnervation of the Bouchaud, 1986), and throughout the entire SCO, SCO when the grafts were implanted in the caudal 9 days after birth. The maturation of the synaptic third ventricle (Ueda et al., 1988). However, when the elements in the rat culminates by postnatal day grafts were inserted in the rostral or mid part of the 21 (Marcinkiewicz and Bouchaud, 1986). In the cat, the third ventricle, no signs of SCO reinnervation by sero- serotonergic innervation of the SCO starts during the tonergic fibers were observed. In the experiments with third postnatal week and is completed 3 months after successful reinnervation, serotonin fibers seem to re- birth (Matsuura et al., 1989). place the non-monoaminergic innervation of the SCO. There is a guidance of the outgrowing serotonin fibers Origin of the Serotonergic Innervation along the ventricular walls to finally innervate the Experiments performing electrolytic lesions have re- basal portion of the SCO. Voutsinos et al. (1994) also vealed that the serotonergic innervation of the rat SCO reported the ability of grafted fetal raphe neurons to 522 A.J. JIME´ NEZ ET AL.

Figs. 1–5. Frontal sections of the SCO immunostained according 10J strain. A dense plexus of immunoreactive fibers occupies the basal to the peroxidase-antiperoxidase method with an antiserum against portion of the SCO (arrows) and the neighboring ependymal surface serotonin (5-HT, Sigma, Spain, S-5545, diluted 1:2,000). Fig. 1. Vi- (open arrow in Fig. 2). Note many labeled cells and fibers in the deep bratome section of the adult rat SCO. Note a dense immunoreactive pineal organ (star in Fig. 2). Fig. 2, ϫ200; Fig. 4, ϫ300 (Jime´nez et al., plexus along the basal portion of the SCO cells (arrows). In the unpublished results). Fig. 3. Detail of Figure 2 showing fibers pene- adjacent ependyma, labeled fibers lie on the apical surface of the trating the secretory ependyma (arrows). ϫ430. Fig. 5. Frozen section ependymal cells (open arrows). ϫ250 (Jime´nez et al., unpublished of the bovine SCO showing labeled fibers in the basal portion of the results). Figs. 2–4. Vibratome sections at rostral (Figs. 2, 3) and mid secretory ependyma (open arrow), some of them penetrating the (Fig. 4) levels through the SCO of a 20-day-old mouse of the C57BL/ ependymal layer (arrows). ϫ480 (Jime´nez et al., unpublished results). INNERVATION OF THE SCO 523 neoinnervate the SCO depending on graft localization. and electron-microscopic levels (Figs. 8–11). In addi- These authors demonstrated specificity in the recogni- tion, the SCO cells were immunopositive with an anti- tion mechanisms between the serotonergic neurons serum against the ␤2 subunit of the GABAA receptor. and their targets. Moreover, whereas the restored su- Recently, responses to GABA treatment have been praependymal plexus contained both serotonin and found in the SCO of the catfish Clarias batrachus GABA, those fibers reinnervating the SCO contained (Saha et al., 2000). After an intracranial administra- only serotonin. At the electron microscopic level, syn- tion of GABA, SCO ependymal cells and Reissner’s aptic contacts were present in the SCO ependyma and fiber showed loss of immunoreactivity to an antiserum absent in the classical ependyma, as occurs in the nor- against calcitonin. This response was blocked by pre- mal animal. All these facts indicate a selectivity of the treatment with bicuculline, a GABAA receptor antag- recognition mechanisms between the serotonergic neu- onist. In teleost species such as Carassius auratus, rons and their targets. Liza aurata, and Coho salmon the SCO lacks of synap- Grafting of different embryonic metencephalic seg- tic contacts (see below). It is not known whether a ments into the third and fourth ventricles of rats has neural input exists in the catfish SCO but, as discussed shown that reestablishment of the serotonergic inner- above, the CSF may be an alternative route for neuro- vation depends on the fetal raphe portion grafted (Di- transmitters to reach the SCO. dier-Bazes et al., 1997). In these experiments, when segments from the medial part of the metencephalon Origin of the GABAergic Innervation were grafted in the vicinity of the habenular recess, As has been mentioned above, Gamrani et al. (1981) against the posterior commissure, they produced dense reported that some of the GABAergic terminals in the plexusses of fibers at the basal pole of SCO ependymo- rat arise from the mesencephalic raphe nuclei. The cytes. other terminals that survive lesions of the raphe nuclei In a recent study, the SCO was grafted into the with injection of 5,7-DHT are of unknown origin. lateral ventricle of the rat (Rodrı´guez et al., 1999). The In Rana perezi, some neurons in the pineal organ and fact that these grafts were not reinnervated confirm fibers in the pineal tract have been labeled with an the view that the location of the SCO is crucial for antibody against GABA (Jime´nez et al., 2000). This targeting its innervation. fact suggests the possibility of an implication of GABA in the pineal-SCO innervation (see below). GABAERGIC INNERVATION Radioautographic studies carried out in rats after Role of the Serotonergic Innervation on the intraventricular injection of tritiated ␥-aminobutyric GABA Uptake by Subcommissural Organ Cells acid (3H-GABA), together with the measurement of the Data reporting the role of serotonin on the accumu- enzyme glutamate decarboxylase (GAD), and the im- lation of GABA by the SCO cells suggest the existence munocytochemical detection of GAD, have shown that of two complementary effects, one related with matu- the labeled amino acid and GAD are present in numer- ration and differentiation, leading to a control of the ous nerve terminals contacting the SCO and within expression of phenotypic characteristics, and other of some of the secretory ependymocytes (Gamrani et al., neuromodulation of the secretory process in differenti- 1981; Weissmann-Nanopoulos et al., 1983). In the SCO ated SCO ependymocytes (Didier-Bazes et al., 1993, ependymocytes, labeling was mainly observed in the pages 461–467, this issue). There is evidence suggest- nucleus, and for this reason it has been suggested that ing that during development, serotonergic terminals GABA acts in the genomic transcription giving rise to induce the expression of GABA transporters in the some metabolic modifications (Didier-Bazes et al., SCO ependymocytes. In the newborn rat, no uptake of 1993, pages 461–467, this issue). Gamrani et al. (1981) 3H-GABA took place before the onset of serotonergic found two types of radiolabeled terminals. Some of innervation (Didier-Bazes et al., 1989). However, the them contained round clear vesicles and large granular 3H-GABA accumulation increased with the appearance vesicles making synaptic contact with the SCO. The of serotonergic terminals in the rat SCO. In addition, others showed only clear vesicles and were found dis- there is an absence of GABA accumulation in the rat persed throughout the SCO. The first type of terminals SCO after destruction of the serotonergic innervation disappeared after lesions of the raphe nuclei with in- with 5,7-DHT at birth (Didier-Bazes et al., 1991). Sup- jection of 5,7-DHT and, therefore, would be the seroto- porting these observations, it has been described that nin-containing terminals innervating the basal portion the SCO of the newborn semi-desert rodent Meriones of the SCO. shawi is capable of GABA uptake and is also inner- We have studied the GABAergic innervation in the vated by serotonin at this age (Laalaouli et al., 1996). rat and bovine SCO under the light microscope by This GABA uptake decreased and disappeared from means of immunocytochemical detection of GAD and the second week of age on coinciding with an increase GABA. Whereas numerous labeled fibers were seen in of the serotonergic innervation. close contact with the secretory ependyma in the rat Other experimental studies have been carried out in (Fig. 6), they were not present in the ependymal layer adult animals, with mature SCO ependymocytes. Gam- of the bovine SCO (Fig. 7). Besides, some groups of rani et al. (1981) found that, after 5,7-DHT treatment bovine SCO ependymocytes were labeled with the GAD or extensive lesions of the mesencephalic raphe nuclei antibody. of the adult rat, the SCO remained strongly radiola- We have reported a GABAergic innervation in the beled. Besides, inhibition of serotonin synthesis with SCO of the frog Rana perezi (Jime´nez et al., 2000). In parachlorophenylalanine increased the rate of 3H- this study, fibers immunolabeled with antibodies GABA accumulation (Didier-Bazes et al., 1989, 1993). against GABA and GAD establishing synapse-like con- Supporting these results, the neurotoxic destruction of tacts with the SCO ependyma were shown at the light- serotonergic innervation with 5,7-DHT in Meriones 524 A.J. JIME´ NEZ ET AL.

Fig. 6. Vibrating microtome fron- tal section of the rat SCO immuno- stained with an antiserum against GABA. Many labeled fibers form a plexus beneath the basal portion of the SCO ependymal cells (arrows). For procedure see Jime´nez et al. (2000). ϫ780 (Jime´nez et al., unpub- lished results). Fig. 7. Frontal frozen section of the bovine SCO immunostained ac- cording to the peroxidase-antiperoxi- dase method using a monoclonal anti- body against GAD-65 (GAD-6; diluted 1:50). Note labeled fibers (arrows) in the posterior commissure and groups of labeled ependymal secretory cells (open arrows). ϫ350 (Jime´nez et al., unpublished results). INNERVATION OF THE SCO 525

Figs. 8–11. Ultrastructural details of nerve endings immunola- a labeled ending showing synaptic vesicles (arrow). Fig. 11. Detail of beled for GABA in the frog SCO. Figs. 8,9. Labeled endings (arrow in a labeled ending (star) near a non-labeled one containing large elec- Fig. 8) contact basal processes of ependymal secretory cells (stars in trodense vesicles (arrow). Fig 8 ϫ42,200; Fig. 9 ϫ60,000; Fig. 10 Figs. 8 and 9). Unlabeled endings are also present. Fig. 10. Detail of ϫ84,500; Fig. 11 ϫ70,000 (Jime´nez et al., unpublished results). 526 A.J. JIME´ NEZ ET AL. shawi led to the reappearance of GABA accumulation (RIA) techniques allowed detection of the presence of into the SCO (Laalaouli et al., 1996). It has been de- several neuropeptides that could provide a neural scribed an absence of GABA accumulation in the adult input to the SCO, such as: luteinizing hormone-re- rat SCO, lacking serotonergic innervation, after graft- leasing hormone (LH-RH) by means of RIA in the rat ing the SCO into the fourth ventricle (Didier-Bazes et (Kizer et al., 1976) and by immunocytochemistry in al., 1991, pages 461–467, this issue). the dog (Matsuura and Sano, 1987); vasopressin us- ing RIA in the rat and rabbit (Dogterom et al., 1979), NORADRENALINE AND DOPAMINE and immunocytochemistry in the rat (Buijs and Pe´- There is evidence of a low concentration of noradren- vet, 1980; Knigge and Schock, 1993) and dog (Mat- aline and dopamine in the SCO (Palkovits, 1987). In suura and Sano, 1987); arginine vasotocin with RIA addition, some fibers presumably containing noradren- in the rabbit (Dogterom et al., 1979; Rosenbloom and aline, detected by immunocytochemistry using anti- Fisher, 1975); oxytocin using RIA in the rat and bodies against tyrosine hydroxylase (TH) and dopa- rabbit (Dogterom et al., 1979) and also with immu- mine-␤-hydroxylase (DBH), have been observed in the nocytochemistry in the rat (Buijs and Pe´vet, 1980) hypendyma of the rat SCO (Balaban et al., 1994). Thus, and dog (Matsuura and Sano, 1987); oxytocin-neuro- Balaban et al. (1994) proposed this modest noradren- physin by means of immunocytochemistry in the ergic input in the rat as a candidate for an excitatory snake Natrix maura (Ferna´ndez-Llebrez et al., 1987; mechanism in contrast with the inhibitory effect pro- Figs. 17, 18); mesotocin in Natrix maura (Ferna´ndez- posed by Bouchaud (1979) for the massive serotonergic Llebrez et al., 1987); somatostatin with immunocy- innervation. tochemistry in the rat (Pelletier et al., 1975); sub- Using light microscopic immunocytochemistry with stance P in the rat (Ho¨kfelt et al., 1978; Knigge and an antibody against TH in the bovine SCO, we have Schock, 1993) and the frog Rana perezi (Fig. 19); observed numerous fibers that could be noradrenergic ␣-neoendorphin using RIA in the rat (Zamir et al., or dopaminergic in nature (Fig. 12). These immunola- 1984); galanin using immunocytochemistry in the beled fibers were in contact with the basal portion of lamprey Lampetra fluviatilis (Jime´nez et al., 1996; the secretory ependyma of the SCO and some pene- Fig. 20), in the turtle Mauremys caspica (Jime´nez et trated into it. This pattern of innervation is very sim- al., 1994; Fig. 21) and in the frog Rana perezi (Figs. ilar to that observed for the serotonergic fibers of the 22, 23); and corticotrophin releasing factor (CRF) in bovine SCO. the rat by means of immunocytochemistry (Knigge In Rana perezi, abundant immunoreactive fibers in and Schock, 1993). In the rat, Knigge and Schock the vicinity of the SCO have been described by use of an (1993) showed the presence of receptors for CRF in antibody against dopamine, but only very few of them the SCO using polyclonal antisera containing anti- were in close contact with the secretory ependyma idiotypic antibodies. (Jime´nez et al., 2000; Fig. 13). However, synaptic con- When the SCO of different vertebrate species is stud- tacts with the SCO basal processes should not be dis- ied under the electron microscope it is possible to detect carded. On the other hand, using an antibody against fibers containing large granular vesicles (Bouchaud, noradrenaline, only a few immunoreactive fibers were 1993; Ferna´ndez-Llebrez et al., 1987; Oksche and Vau- detected near the SCO, although the observation of pel-von Harnack, 1965; Rodrı´guez, 1970), that could be contacts with the ependymal layer of the SCO was of the peptidergic type and could contain any of the frequent (Jime´nez et al., 2000; Fig. 14). In the turtle cited molecules (Fig. 18; see also Fig. 30). Mauremys caspica, we have observed under the elec- tron microscope the presence of terminals with abun- NEURAL CONNECTIONS OF THE dant round clear and few large dense core vesicles SUBCOMMISSURAL ORGAN WITH THE resembling monoaminergic terminals (Fig. 15). PINEAL COMPLEX AND THE CIRCADIAN TIMING SYSTEM NITRIC OXIDE On several occasions, it has been suggested that the In Rana perezi, we have investigated the presence of spatial proximity between the pineal organ and the fibers that could use nitric oxide as a signal by aplying SCO could imply some functional relationships (Ok- the histochemical detection of the neuronal enzyme sche and Korf, 1993). The presence of abundant pine- NADPH-diaphorase, considered a nitric oxide synthase alofugal fibers in lower vertebrates coursing near the (Dawson et al., 1991; Hope et al., 1991). We found two SCO has led to the proposition of a pineal neural input types of labeled fibers in the proximity of the SCO (Fig. to the SCO. In addition, the existence in teleosts and 16). Thick smooth fibers run near the secretory cells amphibians of a direct communication between the and thin fibers formed a plexus close to the SCO pineal lumen and the third ventricle suggests the pos- ependyma. In the pineal tract, a few labeled smooth sibility that any substance with possibilities to be re- fibers, coursing over the SCO, were also detected. In leased into the CSF from the pineal organ, as for ex- the rat, it has been reported that some NADPH-diaph- ample serotonin or melatonin, could reach and influ- orase-positive fibers entered the SCO area coming from ence the SCO activity. labeled neurons distributed throughout the dorsal cen- A tracing study carried out in the silver lamprey tral gray (Knigge and Schock, 1993). Ichthyomyzon unicuspis at the light-microscopic level has shown pineal fibers coursing among the SCO cells NEUROPEPTIDES (Puzdrowski and Northcutt, 1989). However, according Legait and Legait (1956), with the use of Gomori to Ya´n˜ ez et al. (1993), a clear relationship between the stain, described the presence of peptidergic fibers traced pineal fibers and the SCO did not exist in the among the SCO ependymal cells of reptiles. The use larval sea lamprey Petromyzon marinus. In different of immunocytochemical and radioimmunoassay teleost species, a tight spatial relationship between the INNERVATION OF THE SCO 527

Fig. 12. Frontal frozen section of the bovine SCO immunostained with a polyclonal antiserum against tyrosine hydroxylase (TH, Pe-freez Biologicals, P40101-0, Ro- ges, AK, dilution 1:1,000). Immu- noreactive fibers were mainly lo- cated in the basal portion of the ependymal layer. ϫ500 (Jime´nez et al., unpublished results). Figs. 13, 14. Frontal vibratome sections of the frog SCO immuno- stained with antisera against do- pamine (DOPA, Fig. 13) and nor- adrenaline (NOR, Fig. 14). Dopa- mine fibers are numerous in the vicinity of the SCO and few fibers containing noradrenaline can be observed close to the secretory SCO cells (arrows in Fig. 14). Fig. 13, ϫ240; Fig. 14, ϫ260 (Jime´nez et al., unpublished results). Fig. 15. Ultrastructural detail of the SCO of the turtle Mauremys caspica showing a nerve ending (N) containing clear (arrow) and dense-core round-shaped vesicles (arrowhead). ϫ53,100 (J. Pe´rez and P. Ferna´ndez-Llebrez, unpub- lished results). Fig. 16. Vibratome frontal sec- tion at a mid rostrocaudal level of the frog SCO stained for NADPH- diaphorase activity (DIAPH). Thick (open arrow) and thin (ar- rows) labeled fibers are located close to the basal region of the se- cretory ependymal cells. ϫ150 (Jime´nez et al., unpublished re- sults).- pineal tract and the SCO has been found (Puntius horseradish peroxidase (HRP) in the goldfish Caras- sophore, Sathyanesan and Sastry, 1982; Gasterosteus sius auratus has demonstrated that pineal fibers do not aculeatus, Ekstro¨m and van Veen, 1983, Ekstro¨m, make synaptic contacts with the SCO, in spite of pen- 1984; Carassius aureatus and Anguilla anguilla, Ek- etrating the secretory ependyma (Pe´rez-Fı´gares et al., stro¨m and van Veen, 1984; Carassius auratus,Pe´rez- 1993; Jime´nez et al., 1993; Figs. 25, 26). Other unpub- Fı´gares et al., 1993, Jime´nez et al., 1993, 1995, Fig. 24). lished ultrastructural observations in teleosts such as Nevertheless, an ultrastructural tracing study using Liza aurata (observation by the authors) or Coho 528 A.J. JIME´ NEZ ET AL.

Fig. 17. Frontal paraffin section of the snake SCO immunostained Fig. 18. Ultrastructural view of the snake SCO showing many with an antiserum against bovine oxytocin-neurophysin (OXI). Labeled nerve fibers filled with neurosecretory granules (arrows) among the profiles (arrows) penetrate the basal and apical portions of the SCO secretory cells of the SCO. X10,500 (unpublished result by J. Pe´rez cells and in the pineal organ (open arrow). ϫ130 (J. Pe´rez and P. and Ferna´ndez-Llebrez, unpublished results).- Ferna´ndez-Llebrez, unpublished results). salmon (C.R. Yulis, personal communication) indicate to the conclusion that light/darkness conditions hardly the lack of innervation in the teleost SCO. These re- influence the secretory activity of the SCO cells in sults raise the question of how the activity of the teleost Rana temporaria but only slightly in Rana esculenta SCO is controlled. A hypothetical way is through com- (Diederen, 1972, 1973). pounds, such as serotonin, that could be released into To see the influence of light on the SCO, complex the CSF in the paraventricular and posterior recess experimental studies have been carried out in Rana organs (Meurling and Rodrı´guez, 1990), or even as esculenta and Rana temporaria. These experiments mentioned above, from the pineal organ. To prove this, consisted in the separate or combined elimination of the presence of suitable receptors in the apical surface the frontal organ, the pineal organ, and the lateral eyes of the SCO must be demonstrated. (Diederen, 1975), and the use of different light wave- Pineal fibers coursing near the SCO have also been lengths (Vullings et al., 1983). In these studies, evi- found in frogs (Diederen, 1970, 1973; Eldred et al., dence has been obtained indicating that light appar- 1980; Jime´nez et al., 1995; Mautner, 1965; Oksche, ently exerted an inhibitory influence on the growth 1954, 1955, 1956, 1962; Paul, 1972; Paul et al., 1972; rate of Reissner’s fiber. When all of these light-percep- Wake et al., 1974; Fig. 27). On the other hand, at tive organs were eliminated, a clearly higher rate of variance to the situation in teleosts, it is possible to Reissner’s fiber growth was found in light-adapted an- detect ultrastructurally synaptic contacts with the imals than in dark-adapted animals. Diederen and SCO of toads (Murakami and Tanizaki, 1963; Rodrı´- Vullings (1993) have hypothesized that the pineal com- guez, 1970), and the frogs Rana esculenta (Oksche and plex and the lateral eyes exert a spontaneous stimula- Vaupel-von Harnack, 1965) and Rana perezi (Jime´nez tory influence on the SCO in darkness, that light may et al., 1993). compensate and, thus, the secretory activity of the SCO Some studies have been carried out on the influence appeared to be independent of the actual conditions of of light and darkness on the SCO activity of frogs. light and darkness. Light could affect the SCO through photoreceptor or- In brief, the existence of an SCO neural input from gans such as the pineal complex and the lateral eyes. the pineal complex occurs in frogs. The ultrastructural The measurement of several parameters such as the tracing of pineal fibers in Rana perezi using HRP has amount of aldehyde-fuchsin positive material, nuclear shown synapse-like contacts with secretory cells of the volume, and incorporation rate of 35S-cysteine have led SCO (Jime´nez et al., 1993; Figs. 28, 29). Besides, the INNERVATION OF THE SCO 529

Figs. 19–23. Figure legend on next page 530 A.J. JIME´ NEZ ET AL.

Fig. 24. Frontal paraffin section of the goldfish SCO showing HRP- Figs. 25, 26. Ultrastructural demonstration of HRP-labeled pineal labeled pineal fibers immunostained with anti-HRP and intensified nerve endings in the goldfish SCO. Fig. 25. HRP labeled myelinated with silver-methenamine. The SCO and Reissner’s fiber (star) are also (arrow) and unmyelinated (open arrow) pineal axons, occupy the immunostained without intensification using an antiserum against pineal tract just over the SCO cells. Fig. 26. A group of labeled bovine Reissner’s fiber-glycoproteins. Traced pineal nerve fibers are in unmyelinated axons (arrow) penetrated deep among the SCO cells but close contact with the SCO ependyma (arrow). ϫ380 (Jime´nez et al., no nerve endings can be identified. Fig. 25, ϫ4,500; Fig. 26, ϫ18,300 unpublished results). (Jime´nez et al., unpublished results). observation of non-labeled terminals of unknown origin nucleus of the rat allowed tracing of the efferent pro- in the same cells (Figs. 29, 30) supports a multiple jections toward the posterior commissure, some of innervation of the frog SCO. them penetrating into the SCO ependymal layer The injection of Phaseolus vulgaris-leucoagglutinin (Mikkelsen, 1994). This result and the fact that the into the intergeniculate leaflet of the lateral geniculate intergeniculate leaflet of the lateral geniculate nucleus also innervates both the pineal organ and the supra- chiasmatic nucleus have led to the suggestion that the rat SCO could be influenced by the circadian timing Fig. 19. Vibrating microtome frontal section of the frog SCO immu- system (see Miranda et al., pages 541–551, this issue). nostained with an antiserum against substance P (SP, RPN 1572, Amersham International, Buckinghamshire, UK, diluted 1:400) ac- THEORY FOR A MECHANORECEPTOR cording to the PAP method. Many labeled profiles are located in the MECHANISM OF NEURAL CONTROL vicinity of the SCO but not in the organ proper. ϫ180 (Jime´nez et al., unpublished results). A special system of CSF-contacting neurons containing urotensin II-peptide has been described in fish (Yulis and Figs. 20, 21. Paraffin frontal sections of the SCO of a lamprey (Fig. 20) and a turtle (Fig. 21) immunostained with an antiserum against Lederis, 1986, 1988). These cells are exclusively located galanin (GAL). Note labeled fibers (arrows) contacting the SCO in the midline of the central canal wall. The characteris- ependyma in the lamprey. In the turtle, many galanin-immunoreac- tics of these neurons correspond to those described by tive fibers border the SCO. Fig. 20, ϫ330; Fig. 21, ϫ300 (Jime´nez et Vigh and Vigh-Teichman (1973) and Vigh et al. (1977), al., unpublished results). showing an apical dendrite with an atypical cilium and Figs. 22, 23. Details of sections obtained in a vibrating microtome sterocilia touching Reissner’s fiber. In addition, soma- at mid (Fig. 22) and rostral levels (Fig. 23) of the frog SCO, stained tostatinergic CSF contacting-neurons are found at other with an antiserum against galanin (GAL; for immunocytochemical procedure see Jime´nez et al., 1994, 1996). Immunopositive profiles sites of the central canal wall. A feedback mechanism (arrows) contact and penetrate among the SCO cells. Fig. 22, ϫ360; based on a mechanoreceptor theory has been proposed Fig. 23, ϫ390 (Jime´nez et al., unpublished results). (Yulis et al., 1990). These cells could test Reissner’s fiber INNERVATION OF THE SCO 531

Fig. 27. Vibrating microtome frontal section of the frog SCO show- (star). Fig. 29. Detail of labeled synaptic contacts displaying weak ing HRP-labeled pineal nerve fibers in close contact with the SCO. (open arrow) and intense reactions (star). Note a non-labeled profile ϫ300 (Jime´nez et al., unpublished results). containing round-shaped and large granular vesicles (arrow). Fig. 30. Detail of unlabeled synapse-like contacts displaying round clear ves- Figs. 28–30. Ultrastructural demonstration of HRP-labeled pi- icles (open arrow) and granular vesicles (arrow). Fig. 28, ϫ2,550; Fig. neal nerve endings in the frog SCO. Fig. 28. Panoramic view showing 29, ϫ15,500; Fig. 30, ϫ32,400 (E.M. Rodrı´guez and A.J. Jime´nez, labeled synapse-like contacts (arrows) among the basal portions of the unpublished results). SCO cells. Note weakly labeled myelinated fibers in the pineal tract 532 A.J. JIME´ NEZ ET AL. and transmit information by their axons to a region that Didier-Bazes M, Voutsinos B, Aguera M, Peyron C, Akaoka H, Belin innervates or influences the SCO activity. MF. 1997. Specific potentialities of embryonic rat serotonergic neu- rons to innervate different periventricular targets in the adult CONCLUDING NOTES brain. J Comp Neurol 382:29–45. Diederen JHB. 1970. The subcommissural organ of Rana temporaria The SCO of mammals is richly innervated by seroto- L. A cytological, cytochemical, cyto-enzymological and electronmi- nergic fibers and poorly innervated by several other croscopical study. Z Zellforsch 111:379–403. Diederen JHB. 1972. Influence of light and darkness on the subcom- neuronal systems. However, this latter type of neuro- missural organ of Rana temporaria L. A cytological and autoradio- nal input may be of a high functional significance after graphical study. Z Zellforsch 129:237–255. the findings of Gonza´lez et al. (1999) that the ependy- Diederen JHB. 1973. Influence of light and darkness on secretory mocytes of the bovine SCO are functionally coupled activity of the subcommissural organ and on growth rate of Reiss- ner’s fibre in Rana esculenta L. A cytological and autoradiographi- through gap junctions. cal study. Z Zellforsch 139:83–94. On the other hand, the possibility has to be consid- Diederen JHB. 1975. A possible functional relationship between the ered that neurotransmitters and/or neuropeptides may subcommissural organ and the pineal complex and lateral eyes in reach the SCO cells via the CSF, and participate in the Rana esculenta and Rana temporaria. Cell Tissue Res 158:37–60. Diederen JHB, Vullings HGB. 1993. Dynamic aspects of the secretory functional control of these cells through receptors lo- process in the amphibian subcommissural organ. In: Oksche A, calized at the apical plasma membrane, as it has been Rodrı´guez EM, Ferna´ndez-Llebrez P, editors. 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