Horseradish peroxidase study of intact or deafferented infundibular complex in Coturnix quail

HORSERADISH PEROXIDASE STUDY DEAFFERENTED INFUNDIBULAR COTURNIX QUAIL

OF INTACT OR COMPLEX IN

J. OLIVER,C. BOUILL~,S. HERB& and J. D. BAYL~ Laboratoire de Physiologie G&&ale. Universite Montpellier II. France Abstract-Horseradish

peroxidase was injected in the dorsal part of the infundibular complex and demonstrated histochemically in brain sections. In intact quail, cells labelled with peroxidase reaction product were found in the hippocampus, septum, lobus paraolfactorius, dorsal thalamus, preoptic and suprachiasmatic areas. Neither extrahypothalamic nor suprachiasmatic labelling could be detected after neural isolation of the basal hypothalamus, whereas testes growth was still induced by environmental photostimulation. Therefore. the photosensitive and gonadostimulating capacities of the hypothalamic island do not seem to depend upon uncompleted deafferentation of the hypothalamus.

THE EFFECTSof hypothalamic deafferentation on the photosexual reflex in male quail are controversial. Results from our laboratory (OLIVER, HERBUT~ & BAYL~,1973) indicated that photic gonadostimulation was maintained in hypothalamic deafferented quail. Conflicting data have been presented. Total deafferentation of the tuberal region completely blocked testicular growth (DAVIES & FOLLETT, 1974; WADA, 1974) and rise in plasma LH (DAVIES& FOLLE~, 1975) in birds reared in long daily photoperiod. It was therefore necessary to control whether or not all neural afferents to the infundibular complex were interrupted after hypothalamic deafferentation. Since the horseradish peroxidase technique is currently used to identify the cells of origin in various neuronal systems (KRIS~NX~~N, OLS~ON& SJ~TRAND, 1971; LA VAIL & LA VAIL, 1972; WARR, 1973) the present study was undertaken to determine the precise origin of neural projections to the infundibular complex and to detect any uninterrupted fibres allowing for the maintenance of the photoinduced testicular growth. EXPERIMENTAL

PROCEDURES

Male quail were reared under short daily photoperiods (6 h of light from 09.00 to 15.00) to prevent gonadal development. At 6 weeks of age, testes quiescence was controlled through intercostal laparotomy. and a surgical cut was placed around the hypothalamus. The bird was anesthetized with a mixture of chloral hydrate and sodium pentobarbitone (Equi-Thesin; Jensen Salsbery, Kansas City. MO, 0.25 ml/l00 g body weight) and the head held in a stereotaxic frame (David Kopf 900). After retracting the skin and trepanning the skull, the knife (height: 2.5 mm; diameter: 2.5 mm) was medially lowered, the blade facing posteriorly. to the ventral coordinate of the posterior hypothalamus (A) +3.0. (V) 0.0: BAYL~, RAMAIX & OLIVER (1974). The stcreotaxic holder was positioned at an angle of 30‘ to the vertical axis in the rostro-caudal plane in order to obtain a symmetrical anteroposterior position of the blade with respect to the hypothalamic region. _-___ Ahhrwiutim~

HRP.

horseradish

peroxidase.

Rotation

of the knife by 360” achieved

la). Immediately

a complete

cut (Fig.

after surgery, the birds were transferred

to long days (I8 h of light from 06.00 to 24.00) for 2 weeks. Horseradish peroxidase (HRP: type VI, Sigma Chemical Co.) was administrated 6 h before the autopsy. During a 30min interval. 0.1 ~1 of HRP solution (5 mg in IO pl saline) was infused. HRP was stereotaxically injected (coordinates: (A) +4.2. (V) +0.2. (L) -0.3) through a 1 ~1 Microser-Hamilton 7001 N syringe and a micropipette of 50 pm, in the dorsal part of the infundibular complex (Fig. la) under Equi-Thesin anaesthesia. Six hours later. the birds were killed and perfused with 250ml of the fixative of KARNOVSKY(1965). The brains were post-fixed and serially cut on a freezing microtome. Sections of 40 pm were reacted with 3.3’-diaminobenzidine (30 min) and hydrogen peroxide (30 min) (LA VAIL, WINSTON & TISH. 1973) and stained with toluidine blue. HRP treatment was applied to intact quail following an identical process (Fig. ?a).

RESULTS Deafferented quail were polydipsic but no block to gonadal growth occurred. Body weight was slightly decreased in operated birds although they were fed through an oesophageal tube for I week after surgery (Table I). The dark reaction product at the injection site was visible macroscopically in sections reacted for peroxidase. The microscopic appearance of the injection site in controls is shown in Fig. 2b. HRP was injected in the region of the nucleus posterior medialis hypothalami. The regions where cells labelled by retrograde transport of protein were found are indicated in Fig 2a. HRP-positive neurons are shown in telencephalic regions, namely hippocampal, septal and paraolfactive structures, in Fig. 3. Labelled cells lying in diencephalic regions are illustrated in Fig. 4: they occurred in the dorsal thalamic region. preoptic area and suprachiasmatic nucleus. Soma, primary dendritic processes and the axonal process are loaded with HRP reaction product, Labelled telencephalic neurons are generally multipolar. diencephalic cells

989

appear rather ovoid-shaped. After hypothalamic deafferentation the reaction product was also clearly visible at the site of injection. The spread of HRP appeared to cover up the whole dorsal infundibular region. Its extracellular diffusion stretched to the surgical boundary but was restricted to the inside of the hypothalamic island. In deafferented birds. no extrahypothalamic cellular labelling could ever have been detected in any central nervous structure where HRP retrograde transport was observed in controls, i.e. hippocampus, septum nuclei, lobus paraolfactorius. dorsal thalamus and preoptic area. The suprachiasmatic nucleus was free of any HRP labelling.

DISCUSSION

We found again (cf. OLIVER et al., 1973). in this experiment that complete deafferentation of the hypothalamus did not suppress testes enlargement in photostimulated quail. Additional precautions indeed were taken against an eventual incomplete neural isolation, especially through the 30” anterior angulation which was used here to lower the knife. Whereas the vertical coordinate of the extremity of the blade for its sagittal posterior position was at 0.0, it was at - 1.0 for its sagittal anterior position. Thus the blade passed immediately behind the optic chiasma and markedly lower than the supraoptic decussation. Control experiments were carried out under direct visual observation. Forebrain structures were surgically removed (BAYL~, BOUILL~ & OLIVER, 1975) and the knife was stereotaxically lowered and rotated following exactly the same process as in experimental birds. Ante-mortem and post-mortem examinations of the hypothalamic island showed that surgical disconnection was complete. Perhaps more important, these observations showed that a very trivial lateral or rostrocaudal shift of the knife during its stereotaxic placement could lead to significant modification of the surgical result. For example, drastic modification of the actual location of the line of deafferentation was observed whenever the blade passed over the optic chiasma. The very thin strip of metal was bent by this firm tissue and was pushed back toward the region of the nucleus posterior medialis hypothalami. It was therefore quite possible that some part of the gonadotropic regions was reached. It should be recalled that very small electrolytic lesions located in the dorsal part of the infundibular complex (region of the nucleus posterioris medialis hypothalami) are effective in blocking the photosexual reflex (SHARP & FOLLETT. 1969; OLIVER, 1972; STETSON,1972).

Since no cellular labelling could be detected in an) extrahypothalamic nervous structure after placing HRP within the deafferented hypothalamus. it can hit assumed that no intact neural fibers wcrc left at the level of the supraoptic decussation. Therefore it seems that the maintenance of normal testicular dcvelopment in photostimulated quail, although their h!pothalamus was deafferented, was not due to unintcrrupted afferents to the infundibular complex. The intrinsic capacities of the basal hypothalamus of being directly photostimulated have been revealed by the marked testicular enlargement that occurred after implantation of small pellets of radioluminous material in the tuberal or in the dorsal part of the infundihular complex of quail reared under short days; on the other hand, selectively applied photic stimulation to the preoptic and anterior hypothalamic region Induced no testes growth (OLIVER & BAYL~. 1976u.h). The retrograde transport of HRP to suprachiasmatic-preoptic and dorsal thalamic neurons in unoperated birds is particularly interesting since direct retinal projections to these regions have been described in various avian species. Retinal fibrcs enter the nucleus supraopticus in the white-crowned sparrow (OKSCHE. 1970). Thin optic fibres leave laterally from the optic tract to enter the supraoptic area of the drake (BONS & ASSENMACHER.1969). Degenerating fibres appear in the anterior hypothalamus after ocular removal in the chicken (BL~‘~McKF~. 195X: l9hl). Optic nerve section is followed by degeneration of axon terminals located in the suprachiasmatic region of ducks (BONS, 1974) and sparrows (HARTWIG. 1974). At least. autoradiographic investigations evidenced optic nerve terminals in the anterior hypothalamus of the pigeon (MEIER. 1973). On the other hand, COWAN. AL~AMSON& POWELL (1961) described a direct retinal projection to the nucleus lateralis anterior thalami in the pigeon. Using Fink--Heimer methods for degenerating terminals. KAREN & NAL’TA (196X) showed a prominent projection to the whole dorsolateral anterior thalamic complex in the same species. Since the present data indicate direct hypothalamic neural afferents from both preoptic and thalamic origins, it can be proposed that. in unoperated birds. photically induced hypothalamic gonadotropic activity could be mediated via at least three mechanisms: (1) retinal information through the dorsal anterior thalamic complex (part of the ‘thalamofugal visual pathway’ of KARTEN. 1969): (2) retinal signals through suprachiasmatic-preoptic connections; (3) direct deep photostimulation at the infundibular complex lcvcl (OLIVFR & BAYL~. 1976~). It is not possihlc at the

TABLE 1. THE PHOTOSEXUAL REFLEX IN UNOPERATEI) ANI) HYPOTHALAMIC DMAF~FRI'NTII) QUAIL SURIECTED To LONG DAILY PHOTOPERIODS FOR TWO WIXKS

Group Unoperated Deafferented

Number 9 12

Body wt. (g) (mean * Sk.)

Testes wt. (mg) (mean i_ Sk.)

164 + 9 148 f 5

2393 + 228 1990 + 209

FIG. I. (a) Diagrammatic mid-sagittal representation of the complete hypothalamic deafferentation and of the site of in.jection of horseradish peroxidase. AM, nucleus anterior medialis hypothalami; CA, commissura anterior; Cb. cerebellum; CO, chiasma opticum: CP, commissura posterior; DM. nucleus dorsomedialis thalami; DS. decussatio supraoptica; Hp. hippocampus; LPO. lobus paraolfactorius; N3. nervus oculomotorius: P, pineal: POA. area preoptica: S, septum; TSM, tractus septo mesencephalicus: TU. nucleus tuberis; V. ventriculus; 1. site of injection of HRP. (b) Sagittal section showing the outline of deafferentation cut.

FIG. 2. Hypothalamic horseradish peroxidase administration and reaction product in the brain of intact quail. (a) Lateral view from the midsagittal plane showing the injection site (1) and location of hippo), thalamic (T: +), paraolfactive (LPO: *), preoptic (PO: 0) and suprachiascampal (H: A), septal (S: ?? ) labelled neurons. CA. commissura anterior; CO. chiasma opticum; CP. commissura matic (SC: ?? posterior; N3. nervus oculomotorius; TSM, tractus septo-mesencephalicus; TU. nucleus tuberis. (b) Parasagittal section through the hypothalamus showing the injection site (I) and spread of HRP.

FIG

3. Labelled neurons in telencephalic structures. (a. b) Hippocampus; (c. d) Nucleus medialis; (e, f) Lobus paraolfactorius. Magn.: a. c. e: x 580; b, d. f: x 1450.

septalis

FIG. 4. Horseradish peroxldase labelling m diencephalic neurons. (a. b) Nucleus dorsalis thalaml: (e. f) Preoptic area. Magn.: a. c. e; x 580: h. d. f: x 1450 d) nucleus suprachiasmaticus;

(c.

Peroxidase labeliing and hypothalamic deafferentation in quail to speculate on the relative possible role of these three componants. Cells labelled with peroxidase reaction product were previously found in the hippocampal and septal regions after HRP injection in the medial hypothalamus of the pigeon (BOUILL~,RAYMOND& BAYL~. 1977). Their distribution in the present study was quite similar to the regular scattering throughout the hippocampus and the septum found by the latter authors. It is not possible to ascribe a gonadotropic role to such hippocampal and septal infundibular moment

connections. It is perhaps

worth noting that HRP was retrogradely transported to some neurons of the paraolfactory lobe. Possibly this location might correspond to the rhinencephalic

region

which

was photostimulated

di-

0’)

rectly by BENOIT & KEHL (1939) in the drake, resulting in testicular growth. Since the infundibular complex was stereotaxically reached with the stereotaxic holder positioned at an angle of 30” anteriorly, the pipette was largely anterior to the hippocampus and the dorsal thalamus. and posterior to the paraolfactive and preoptic areas. The micropipette passed through the corticoseptalis tract and it was impossible to avoid direct injury to axons of this tract. Hence artifactual labelling of septal neurons could be possible. However. no ventricular or ependymal diffusion of the enzyme was observed. Ackrtoul~~dgr~mrrltsThis work was supported by (‘NRS Grant ERA 85. Thanks are due to Dr CARRION. Laboratoirc de Parasitologle Cornparke. Llniversitb Montpellier 11.

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