A retinal projection to the paraventricular nuclei of the hypothalamus in the Syrian hamster (Mesocricetus auratus)

Brnin Rewarch

Bulktin,

0361-9230187 $3.00 + .OO

Vol. 19. pp. 747-750. ’ Pergamon Journals Ltd.. 1987. Printed in the U.S.A.

RAPID COMMUNICATION

A Retinal Projection to the Paraventricular Nuclei of the Hypothalamus in the Syrian Hamster (Mesocricetus auratus) TIMOTHY Dcpurtmrrlt

G. YOUNGSTROM,

of Psyc~hology

MARK

L. WEISS

NII~ A~c~trrosc~iotc~cProgram,

Received

AND ANTONIO

Michigctn

10 August

Stutr

University.

A. NUNEZ Eust Lrtrlsing, MI 48824

1987

T. G.. M. L. WEISS AND A. A. NUNEZ. A w/inu/ pmjccfiou to the pnrrrl,rntric,rr/rr~ nrrc,/ci cfthc hamstcv (Mesocricetus auratus). BRAIN RES BULL 19(6) 747-750, 1987.-The retinohypothalamic tract of the Syrian hamster (Mc.soc,ric,ctf,.s trrrrcrtrrs) was examined using intraocular injections of horseradish perixodase conjugated to cholera toxin. In addition to the retinal input to the suprachiasmatic nuclei (SCN), a retinal projection to the caudal paraventricular nuclei was identified. This projection may mediate some effects of light upon pineal gland physiology independently of the SCN and the circadian-rhythm generating system. YOUNGSTROM,

h~porhalumus

io

t/w Syricrtz

Paraventricular nucleus Cholera toxin-horseradish

Suprachiasmatic perokdase

nucleus

Retino-hypothalamic

METHOD

Criteria for Evaluating Alternate Transport Mechunisms

Animals and Procedures

and female (n=8)

Pineal gland

(Mrsocricc,tlrs clrrratus; 12&200 g) were obtained from Charles Rivers Laboratories and maintained on a l6:8 L:D cycle. Animals were anesthetized with Equithesin (4.5 ml/kg) and received unilateral intraocular injections (1.5-3.0 ~1) of CT-HRP (0.38-0.4%). The conjugate was prepared from HRP (Sigma Type VI) and cholera toxin (Sigma Prod. No. C9025) in the laboratory of Dr. R. R. Miselis at the University of Pennsylvania using methods previously described [20]. Following survival periods of 12-48 hr, the animals were deeply anesthetized with Equithesin and perfused transcardially using a previously described technique [ 121. Frozen corona1 sections (50 pm) were collected in 0.1 M phosphate buffer (pH 7.4) and subsequently processed using the tetramethylbenzidine protocol of Mesulam [ 121. The sections were arranged into three sets of adjacent serial sections, each set consisting of every third section. All sections were mounted on previously gelatinized slides. Two sets were air dried, cleared in xylene and coverslipped using Permount. One set of tissue was air dried and then counterstained with cresylecht violet prior to coverslipping. The tissue that was not counterstained was evaluated immediately using brightfield and darkfield optics.

A retino-hypothalamic tract (RHT) which projects primarily to the suprachiasmatic nuclei (SCN) has been described in a variety of mammalian species [l3, 14, 161. The SCN are involved in the generation of circadian rhythms and photoperiod dependent seasonal cycles [19,23]. In Syrian hamsters, the paraventricular nuclei of the hypothalamus (PVN) are also part of the neural pathway that mediates photoperiodic responses [10,17]. Retinal input to the region of the hypothalamus between the SCN and PVN has been reported using intraocular injections of unconjugated horseradish peroxidase (HRP). This retinal input outside the SCN was particularly salient in photoperiodic rodents, and in some cases granules of reaction products were seen close to the ventral boundary of the PVN [16,26]. Furthermore, injections of HRP just dorsal to the SCN of hamsters produced retrogradely labelled retinal ganglion cells [ 151. In the present study, we re-examined the retina1 input to the SCN and PVN of the hamster using intraocular injections of HRP conjugated to cholera toxin (CT-HRP). Previous work using rats [ll] has shown that when injected intraocularly, CT-HRP produces more intense labelling of the RHT than that obtained with similar injections of HRP.

Adult male (n=lO)

tract

Syrian

The tissue was examined for evidence of hemal or transsynaptic transport of the CT-HRP. First, the magnocellular

hamsters

747

748

YOUNGSTROM,

FIG. I (A) Photomicrograph showing cholera toxin-horseradish peroxidase (CT-HRP) reaction product in a coronal section (darkfield illumination) through the caudal half of the suprachiasmatic nuclei (SCN). The animal received a 3 J?LIunilateral intraocular injection (L=left eye) of CT-HRP and survived for 48 hr. The ventral 2/3 of each nucleus possessed such a high density of labelling that it appears dark in photomjcrographs. Labelling was observed throughout the anterior-posterior extent of the SCN with the least amount of reaction product found in the dorsomedial part of the nuclei. (B) Camera lucida drawing of the same section after counterstaining with cresylecht violet to show the boundary of the SCN. (Bar for A and B=lOO pm: V-third ventricle; OC-optic chiasm). PVN and supraoptic nuclei (SON) were examined to determine if retrogradely labelled cells were present. In a number of rodent species, magnocellular neurons within these two nuclei are retrogradely labelled when HRP is injected into the systemic circulation [3]. Second, the primary visual cortex was examined for evidence of orthogradely transported CT-HRP. Labelling in this area of cortex would be indicative of trans-synaptic transport in at least one target nucleus of the optic tract [9]. RESULTS

There were no differences between sexes with respect to pattern or intensity of iabelling. The intensity of labelling increased with increased survival time but there were not changes in the pattern of labelling across survival times. No retrogradely labelled cells were found in the magnocellular PVN or SON and no reaction product was observed within primary visual cortex even after the longest survival time (48 hr). As shown in Fig. I, the pattern of labelllng within the SCN was essentially the same as has been reported previously following unilateral intraocular injections of HRP [ 161.

WEISS

AND NUNEZ

FIG. 2. Camera lucida drawings of sections from a representative case that received a unilateral injection of cholera toxin-horseradish peroxidase into the left (L) eye and survived for 48 hr. Each drawing is a composite of labelled fibers observed in six adjacent sections plotted onto tracings of the PVN. The third section of the six was stained with cresylecht violet in order to draw the PVN. Few labelled fibers were located in the anterior magnocellular PVN. Most fibers were observed in and ventral to the posterior subdivisions corresponding to the parvocellular PVN as described for the rat [22]. A few fibers were observed dorsal and caudal to the PVN, and occasionally, fibers were seen as far caudal as the dorsomedial nucleus of the hypothalamus. (Bar=100 ym; A-anterior, P-posterior, R-right, PVN-paravent~~ular nuclei of the hypoth~amus, V-third ventricle).

In addition, labelled fibers were observed dorsal and lateral to the SCN. These fibers became more numerous in the caudal half of the SCN and projected to an area ventral to the medial half of the PVN (Fig. 2). A few fibers along the ventral boundary of the PVN turned dorsally to enter areas analogous to the lateral parvocellular, periventricular and dorsal parvocellular regions of the PVN as described for the rat [223. The heaviest labelling in the PVN was in the ventro-medial portion of the lateral parvocellular subdivision. On the other hand, very few fibers were found within the ma~ocell~ar portion of the PVN. As shown in Fig. 3, the fibers that entered the PVN were thin with enlargements or swellings along their length. DISCUSSION

As previously reported for the Skrian hamster [16] and other mammalian species [13,26], the SCN were found to be the principal terminal nuclei for the retinal input to the hypothalamus. Within the SCN, this input was heaviest in the ventral and dorsolateral portions of the nuclei and was lightest in the dorsomedial area, thus confirming the results of earlier experiments with intraocular injections of HRP [16]. The dist~bution of input from each retina to the two SCN was nearly symmetrical. This symmetry seems to be genus

RETINAL

PROJECTION

TO THE PARAVENTRICULAR

FIG. 3. (A) Photomicrograph of fibers in the paraventricular nuclei of the hypothalamus (PVN; darkfield illumination; Bar=20 pm) labelled after a unilateral intraocular injection of cholera toxinhorseradish peroxidase into the left (L) eye. The labelling appeared as chains of bead-like reaction product connected by thin labelled fibers. The observed enlargements (arrows) are similar to those associated with axon terminals (see [6]). (B) Camera lucida drawing of a coronal section (Bar=100 Frn) through the caudal PVN. The photomicrograph (A) was obtained from the region of the PVN enclosed by the box.

specific since it is present in the Syrian and Turkish hamster [ 16,261, but not in other rodents 114,261. In addition to a retinal input to the SCN, the present results showed that some axons of retinal origin course dorsally through the caudal half of the SCN, leave these nuclei and enter the PVN. Our observations are consistent with previous reports of reaction product dorsal to the SCN after intraocular injections of HRP [16]. However, with the method used here, labelled fibers outside the boundaries of

NUCLEI

the SCN, in contrast to the isolated granules of reaction product seen previously, were easily identified and followed as they entered the PVN. Within the PVN, the fibers showed enlargements usually associated with terminal expansions [6]. Thus, in addition to the SCN, and areas of the lateral [ 11,181 and anterior hypothalamus [ 11,161 the PVN should be considered a terminal site for a direct retino-hypothalamic projection. A similar pattern of labelled fibers within the PVN of rats has been seen after intraocular injections of CT-HRP (J. Levine, personal communication; Youngstrom and Weiss, unpublished observation). However, in the rat relatively less reaction product was seen in the SCN, and fewer labelled fibers were seen dorsal to the SCN and within the PVN. The SCN send efferent projections to the PVN and the sub-PVN area [ 1, 21, 24, 251. However, the observed labelled fibers in these areas after our CT-HRP injections were most likely of retinal rather than of SCN origin. No transsynaptic transport of the tracer was evident, and the pattern of labelling remained constant across different survival times. Furthermore, there was no indication of uptake of the tracer by neurons known to project outside the blood-brain barrier; thus, the reaction product detected in the hypothalamus was likely the result of neural transport of the tracer from the retina. A current model for the neural control of the mammalian pineal gland includes the PVN as relay nuclei between the SCN and the preganglionic sympathetic neurons of the spinal cord [23]. Sympathetic input to the pineal gland is necessary for the expression of a circadian rhythm in pineal serotonin N-acetyltransferase (NAT) activity and melatonin pcoduction, and probably necessary as well for the acute effects of light upon NAT activity and melatonin synthesis (see [2, 7, 81 for reviews). Neurons of the lateral parvocellular PVN are retrogradely labelled following injections of HRP into the spinal cord of hamsters [4,5]. Thus, the direct retinal input to the parvocellular PVN reported here could mediate effects of light upon pineal gland physiology independently of the SCN and the circadian system. Alternatively, cells of the PVN may integrate direct and indirect retinal inputs with circadian signals generated by the SCN and use this information to mediate photoperiod-induced changes in physiology and behavior [23]. ACKNOWLEDGEMENTS The authors thank Dr. R. R. Miselis for supplying the Cholera Toxin-HRP and J. Levine for sharing his unpublished data with us. Supported in part by NIMH Grant MH37877 to A.A.N. and NRSA Postdoctoral Grant NS 08125 to M.L.W.

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750

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