an immunocytochemical mapping of .endorphin (1-27) in the ... · an immunocytochemical mapping of...

Neuropeptides (1996) 30 (3), 261-271 © Pearson Professional Ltd 1996

An immunocytochemical mapping of .endorphin (1-27) in the cat diencephalon

R. CoveSas ~, M. de Le6n ~, J. A. Narv~ez 2, G. Tramu 3, J. A. Aguirre 2, S. Gonz~lez-Bar6n 2

IUniversidad de Salamanca, Facultad de Medicina, Departamento de Biologia Celular y Patologia, Salamanca, Spain 2 UNversidad de Mflaga, Facultad de Medicina, Departamento de Fisiologfa, Mgtlaga, Spain 3 Universit6 de Bordeaux I, Laboratoire de Neurocytochimie Fonctionnelle, CNRS, Talence, France

Summary The distribution of ~-endorphin (1-27) immunoreactive cell bodies and fibres was studied in the diencephalon of the cat using an indirect immunoperoxidase technique. In the thalamus, almost all the immunoreactive fibres were found in the midline region and in nuclei located near the midline, whereas in the hypothalamus fibres containing J3-endorphin (1-27) were visualized extending by the whole structure. The hypothalamus showed a higher density of J3-endorphin (1-27) immunoreactive fibres than the thalamus, as well as immunoreactive cell bodies, since'in the thalamus no I~-endorphin (1-27) immunoreactive neuron was located. The densest network of immunoreactive fibres was observed in the epithalamus (nucleus periventricularis anterior) and in the hypothalamic nuclei arcuatus, hypothalami ventromedialis, suprachiasmaticus, periventricularis hypothalami, hypothalamus dorsomedialis, area hypothalamica dorsalis, hypothalamus anterior, filiformis, hypothalamus posterior and regio praeoptica. In the hypothalamus, a high density of perikarya containing I~-endorphin (1-27) was observed in the nucleus arcuatus and a low density in the nucleus hypothalami ventromedialis. The distribution of J3-endorphin (1-27) immunoreactive fibres and perikarya is compared with the location of other neuropeptides in the cat diencephalon. Our findings reveal that b-endorphin (1-27) immunoreactive structures are widely distributed in the cat diencephalon, suggesting that the peptide might be involved in several physiological functions.

INTRODUCTION

Over the last 9 years the anatomical distribution of several neuroactive substances (peptides and classical neu- rotransmitters) has been described in the cat diencephalon using immunocytochemical techniques. Thus, the presence of peptides belonging to several peptidergic families has been studied: e.g. vasoactive intestinal polypeptide (growth hormone-releasing factor/

Received 15 August 1995 Accepted 22 December 1995

Correspondence to: R. CoveSas, Universidad de Salamanca, Facultad de Medicine, Departamento de Biologia Celular y Patologia, Avda. Campo Charro s/n, 37007 Salamanca, Spain.

peptide histidine isoleucine), ~ substance P and neurokinin A (tachykinin)y neuropeptide ¥ (neuropeptide Y/pancreatic polypeptide) 4,5 and cholecystokinin (gastrin/cholecystokinin). 6 In addition, other peptides that do not fit into the previous groups have also been described in the cat diencephalon such as neurotensin z and somatostatin. ~

It is known that there are three families of opioid peptides in the brain according to the precursors of such peptides: (1) ~-, and 7-endorphin, [~-endorphin (1-31), as well as [3-endorphin (1-27) are produced from pro- opiomelanocortin; (2) methionine-enkephalin, methionine-enkephalin-Arg-Phe, methionine-enkephalin-Arg- Gly-Leu, leucine-enkephalin, bovine adrenal medullary opioid dodecapeptide, peptide F, bovine adrenal medullary opioid docosapeptide, synenkephalin and pep-

261

262 Cove#as et al

fide E from pro-enkephalin; and (3) dynorphin A(1-17), dynorphin A(1-8), leumorphin, a-neo-endorphin, dynorphin B (rimorphin) and [3-neo-endorphin from prodynor- phin. Scarce data are available on the distribution of immunoreactive structures containing opioid peptides in the cat diencephalon. In the feline, it has been only described the distribution of [3-endorphin (1-31) in the hypothalamus 9 and the anatomical distribution of fibres and cell bodies containing methionine-enkephalin in the diencephalon. 1°-12 At present, the only peptides derived from pro-opiomelanocortin studied in the cat CNS were [Mipotropin and [~-endorphin (1-31)? The former, a pro- opiomelanocortin fragment containing [~-endorphin and itself devoid of opiate activity, was described in the brainstem) 3 Thus, no previous information appears to be available in the literature concerning the distribution of fibres and cell bodies containing [3-endorphin (1-27) in the cat diencephalon.

At present, in the CNS of other mammals (e.g. rat and human), the distribution and functional roles of [3-endorphin (1-31) 14-17 are better known that those of cz-, and 7- endorphin and [3-endorphin (1-27). Thus, [~-endorphin (1-31) has been involved in pain, stress, sexual, respira- tory, ageing and feeding mechanisms, as well as modulat- ing the pituitary, the sympathetic nervous system responses and the activities of several central neurotrans- mitters (e.g. dopamine, serotonin and acetylcholine) (for review see ref. 17). Moreover, [~-endorphin (1-31) has been implicated in blood pressure and temperature regulation and has been shown to affect protein and RNA synthesis and adenylate cyclase activityY

Thus, the aim of this work is to know the distribution of immunoreactive structures containing [3-endorphin (1-27) in the cat diencephalon and to compare this distribution with previous studies carried out on the presence of [~-endorphin (1-31) in the diencephalon of other species (e.g. rat, human), as well as with the distribution of several opioid and non-opioid peptides previously described in the cat diencephalon.

MATERIALS AND M E T H O D S

Five male adult cats (2-3 kg body weight), obtained from commercial sources, were used in this study. Each animal was kept in a cage under standardized conditions of light (lights on at 06:00, off at 20:00) and temperature (25°C) and had free access to food and water. The animals remained 10 days in the cage before experiments. Two animals (control) were deeply anaesthetized with ketamine (40-50 mg/kg intraperitoneally), heparinized and perfused transcardially with 500 ml of cold 0.9% NaC1, this pre-rinse followed immediately by the fixative, 3 1 of cold 4% paraformaldehyde, in 0.15 M phosphate- buffered saline (PBS) (pH Z2). The brains were removed,

the diencephalons dissected out and placed in the same fixative for 12 h at 4°C. After this post-fixation, diencephalons were cryoprotected by immersion in increas- ing sucrose baths (10-30%) until they sank. 60-80 gm-thick frozen sections were cut on a cryostat, collected into PBS and kept at 4°C. In general, 6 of 7 sections were used for immunocytochemistry, and the remaining section was stained for Nissl.

Three cats, under deep ketamine anaesthesia, were placed in a stereotaxic apparatus for surgery. Body temperature was maintained at 37°C with a feedback-controlled heating pad. A hole was drilled in the skull and a glass micropipette filled with a saline solution containing colchicine was introduced into the brain. The animals received unilateral intraventricular (lateral ventricle) injections of colchicine (300 gg of the drug diluted in 5 gl of saline solution) in order to enhance the immunoreactivity of cell bodies (the drug acts on microtubule systems and then inhibits axoplasmic transport). Following a 2-day survival time, animals were again deeply anaesthetized. The perfusion and the methodology followed was identical to that described in control animals.

Immunocytochemistry

The free-floating sections were preincubated for 30 rain in 1% normal horse serum in 0.15 M PBS containing 0.3% Triton X-100 in order to enhance the penetration of antibodies. Then, the sections were incubated overnight at 4 ° C in the same buffer containing the anti-I~-endorphin (1-27) antiserum, at the dilution of 1/1000. After a 30-min wash with PBS, the sections were incubated for 1 h with sheep anti-rabbit immunoglobulin coupled to horseradish peroxidase as the second antiserum, diluted 1/250 in phosphate buffer. After washing in PBS (30 rain) and Tris-HC1 buffer (pH Z4) (10 rain), the tissue-bound peroxidase was developed by the 3,3' diaminobenzidine method.

Specificity of the antiserum

The antiserum was raised in rabbits against immunogens prepared by coupling the peptide, synthetic human [~- endorphin (1-27) (purchased from Bachem, Switzerland), to a carrier protein (human serum albumin) with glu- taraldehyde. The specificity of the immunostaining was controlled by: (1) the preabsorption of the first antiserum with synthetic [~-endorphin (1-27) (100 gg per ml of diluted antiserum); (2) omitting the [3-endorphin (1-27) antiserum in the first incubation bath (in both cases, no residual immunoreactivity was found); (3) no significant reduction in the immunolabeUing was found when [~- endorphin (1-27) antiserum was preabsorbed with an excess (10 .7 M) of synthetic [~-endorphin (1-31), a-, and

Neuropeptides (1996) 30(3), 261-271 © Pearson Professional Ltd 1996

Immunocytochemical mapping of fl-endorphin (1-27) in cat diencephalon 263

y-endorphin, [~-lipotropin, adrenocorticotropin hormone, ~-,/3-, and 7-melanocyte-stimulating hormone, methionine-, and leucine-enkephalin, dynorphin A, a-neo-endorphin or dynorphin B; and (4) possible interference by endogenous peroxidases was ruled out by staining some sections beginning with the diaminobenzidine step. No reaction was visualized. The term [3-endorphin (1-27)- like immunoreactive ([3-endorphin-ir) was used to described the staining results in our material. Finally, immunoreactivity was always found in neuronal cells. Non-neuronal cells were devoid of immunoreactivity.

Mapping was carried out according to the stereotaxic atlas of Jasper & Ajmone-Marsan. TM Nomenclature of anatomical structures were taken from this same atlas.

RESULTS

[3-endorphin-ir structures were widely distributed throughout the diencephalon of the cat (Fig. 1). In this Figure, cell bodies containing 13-endorphin (1-27) have been indicated by dots (1 dot = 9 immunoreactive neurons). The density of such immunoreactive neurons has been considered as high (>20 cell bodies/section), middle (10-20 cell bodies/section) and low (<10 cell bodies/section), whereas an attempt was made to grade the density of [3-endorphin-ir fibres into 4 categories: high, moderate, low and single fibres. This was carried out by viewing the sections under light illumination at a constant magnifica- tion with reference to photographs of the defined series of densities. Examples of these ratings are observed in the following figures: high density (Fig. 3D), moderate density (Fig. 3C) and low density (Fig. 3.4). The distribution of ~-endorphin-ir structures presented in Figure 1 is based on the results obtained from both control and colchicine-treated cats.

As shown in Figure 1, in the thalamus, in general, immunoreactive fibres were observed in the midline region but not in the lateral thalamic region, whereas in the hypothalamus the immunoreactivity was found by the whole structure. In general, in the latter diencephalic region the density of ~-endorphin-ir fibres dis- minishes from midline to more laterally located structures. Moreover, the hypothalamus showed a higher density of 13-endorphin-ir fibres than the thalamus, as well as immunoreactive cell bodies, since in the thalamus no/3-endorphin-ir neuron was located. Thus, in the thalamus, except in the nucleus periventricularis anterior that showed a high density of immunoreactive fibres, the other thalamic nuclei showed a low density or single [~-endorphin-ir processes. However, in the hypothalamus, numerous nuclei showed a high or moderate density of immunoreactive fibres, such as hypothalamus posterior, area hypothalamica dorsalis, filiformis, hypothalami ventromedialis, periventricnlaris

hypothalami and hypothalmus anterior. In comparison with control cats, no significant decrecement in the staining intensity of immunoreactive fibres was observed in animals treated with colchicine, as well as there were no significant differences in fibre distribution between both animal groups.

The hypothalamus (mediobasal part) showed a great number of/3-endorphin-ir cell bodies. In both control and colchicine-treated animals, we observed immunoreactive perikarya. However, the number of such neurons is greater in cats treated with colchicine than in control animals, as well as the staining intensity was dramatically enhanced following intraventricular injection of colchicine. Moreover, B-endorphin-ir perikarya are more widely distributed in the hypothalamus of animals treated with colchicine. Thus, in untreated cats, [~-endorphin-ir neurons were observed in the nucleus arcuatus, whereas in animals treated with colchicine, in addition, we observed immunoreactive cell bodies in the nucleus hypothalami ventromedialis. In general, the vast majority of [3-endorphin-ir cell bodies found in the cat hypothalamus were large (perikarya having an average diameter of about 17-25 ~m) and round with short (in general exhibit two dendritic processes) or not visible processes. However, some larger fusfform cells were also found.

Diencephalic-mesencephalic junction

From anteriority (A) 3.0 to A 7.0 (not shown in Fig. 1). At this level no [3-endorphin-ir cell body was observed. However, a moderate density of immunoreactive fibres was found in the griseum centrale, praetectum and in the colliculus superior and a low density in the nuclei interpe- duncularis and commissurae posterioris, in the regions located between both nuclei ruber and between this nucleus and the substantia nigra, below the nucleus ruber and in the substantia reticularis mesencephalica. Single fibres were located in the substantia nigra and in the nuclei of the third nerve, of Darkschewitsch and tuber.

Thalamus

The thalamic nuclei can be grouped, according to topo- graphic criteria, into severn nuclear groups (anterior, posterior, ventral, medial, midline, intralaminar) and complexes (Lateralis posterior-pulvinar, geniculate), as well as in epithalamus and ventral and dorsal thalamus. 19,2° Almost all the immunoreactivity was found in the midline region or in nuclei located near the midline (Fig. 1A-E). Laterally, we only observed immunoreactivity in the nuclei centralis lateralis (Fig. 1B, C), ventralis anterior (Fig. 1C-E), pars ventralis of the corpus geniculatum laterale (Fig. 1,4) and in the corpus geniculatum mediale.

© Pearson Professional Ltd 1996 Neuropeptides (1996) 30(1), 261-271

264 Cove~as et al

H PT S

A g . B AO

~ PVA

\ ~ \ /v, ' I ~ r-RE

• " v "

I L ~ ARC 1 1 . 1 5

CD

• c, ~ ~)r I ~. FX

s o SCH A 1 4

Fig. 1 Distribution of 13-endorphin-ir fibres and cell bodies in frontal planes of the cat diencephalon corresponding to the anteroposterior stereotaxic plane levels from A 7.5 to A 14 of the Jasper & Ajmone-Marsan stereotaxic atlas. 18 Immunoreactive fibres are represented by continuous lines and cell bodies by dots, each dot indicating 9 immunoreactive neurons, The anteriority (A), in mm with respect to the zero stereotaxic point of each section is indicated at the lower right.



Fig. 1 Abbreviations AD: nucleus anterior dorsalis, AHD: area hypothalamica dorsalis, AM: nucleus anterior medialis, ARC: nucleus arcuatus, AV: nucleus anterior ventralis, CA: comrnissura anterior, CD: nucleus caudatus, CI: capsula interna, CL: nucleus centralis lateralis, CM: nucleus centrum medianum, EN: nucleus entopeduncularis, FIL: nucleus filiformis, FX: fornix, GL: corpus geniculatum laterale, GLV: pars ventralis of the corpus geniculatum laterale, GM: corpus geniculatum mediale, GP: globus pallidus, HBL: nucleus habenularis lateralis, HBM: nucleus habenularis medialis, HDM: hypothalamus dorsomedialis, HL: hypothalamus lateralis, HP: hypothalamus posterior, HVM: nucleus hypothalarni ventromedialis, IAM: nucleus interanteromedialis, LD: nucleus lateralis dorsalis, LP: nucleus lateralis posterior, MD: nucleus medialis dorsalis, ML: nucleus mamillaris lateralis, MM: corpus mamillare, NCM: nucleus centralis medialis, NPR: nucleus prothalamicus, PC: nucleus paracentralis, PED: pedunculus cerebralis, PT: nucleus parataenialis, PUL: nucleus pulvinar, PVA: nucleus periventricularis anterior, PVH: nucleus periventricularis hypothalami, R: nucleus reticularis, RE: nucleus reuniens, RH: nucleus rhomboidens, RPO: regio praeoptica, S: stria medullaris, SCH: nucleus suprachiasrnaticus, SM: nucleus submedius, SN: substantia nigra, SO: nucleus supraopticus, SPF: nucleus subparafascicularis, SPT: area septalis, ST: stria terminalis, TO: tractus opticus, VA: nucleus ventralis anterior, VL: nucleus ventralis lateralis, VM: nucleus ventralis medialis, VPL: nucleus ventralis posterolateralis, VPM: nucleus ventralis posteromedialis, ZI: zona incerta,

Immunoreactive fibres The epithalamus (from A 6.0 to A 13.5) showed the high- est immunoreactivity, since a high density of ~-endorphin-ir fibres was observed in the nucleus periventricularis anterior (Fig. 1A-E, Fig. 2A), and a low density in the nuclei habenularis lateralis (Fig. 1A) and habenularis medialis (Fig. 1A). In the midline or central group (from A 8.0 to A 12.5), we observed a low density of immunoreactive processes in the following nuclei: rhomboidens (Fig. 1B-D), reuniens (Fig. 1B-E, Fig. 2B), centralis medialis (Fig. 1B, C), interventricularis and interanteromedialis (Fig. 1D). A low density was also observed in the nuclei of the medial group (from A 6.5 to A 13.0) medialis dorsalis (Fig. 1A-C) and parataenialis (Fig. 1B-E). In the intralarninar group (from A 6.5 to A 11.0), we found a low density of [~-endorphin-ir fibres in the nucleus parafascicularis and single fibres in the nuclei centrum medianum (Fig. 1A) and centralis lateralis (Fig. 1B, C). No immunoreactive fibre was located in the nuclei paracentralis (Fig. 1B, C) and subparafascicularis (Fig. 1.4). Regarding the ventral group (from A Z0 to A 12.5), a low density of fibres containing ~-endorphin (1-27) was found in the nucleus ventralis medialis (Fig. 1 B-D) and single fibres in the nucleus ventralis anterior (Fig. 1C-E), but no immunoreactive fibre was located in the nuclei ventralis lateralis (Fig. 1B-D), ventralis posterolateralis (Fig. 1B-D) and ventralis posteromedialis (Fig. 1A, B). In the posterior group (from A 4.0 to A Z0), single immunoreactive fibres were located in the nucleus limitans, being the nuclei suprageniculatus and posterior devoid of such immunoreactivity. In the lateral

posterior-pulvinar complex (from A 4.0 to A 10.5), we observed single fibres in the first nucleus, as well as in the geniculate complex (from A 3.0 to A 9.0). Thus, we found single fibres in the corpus geniculatum mediale, pars ventralis of the corpus geniculatum laterale (Fig. 1A) and in the corpus geniculatum laterale. The dorsal thalamus (anterior group and nucleus lateralis dorsalis) (from A 7.0 to A 13.0) showed single immunoreactive fibres in the latter nucleus (Fig. 1.4, B), being the anterior group (nucleus anterior ventralis (Fig. 1C-E), nucleus anterior dorsalis (Fig. 1C-E) and nucleus anterior medialis (Fig. 1D, E)) devoid of [3-endorphin-ir fibres. In the ventral thalamus (from A Z5 to A 13.5) a low density of fibres containing [3-endorphin (1-27) was found in the zona incerta (Fig. 1A, B), whereas no immunoreactive fibre was observed in the nucleus reticularis (Fig. 1A-E). Finally, in the nucleus prothalamicus a high density of fibres con- raining [3-endophin (1-27) was observed.

Tracts Single immunoreactive fibres were found in the stria medullaris (Fig. 1A-E) and in the stria terminalis (Fig. 1E).

Immunoreactive cell bodies No cell body containing observed in the cat thalamus.

/3-endorphin (1-27) was

Hypothalamus

This diencephalic structure can be divided, according to tophographic criteria, into several regions: mamillar, periventricular, medial, lateral and preoptic. 21 Structures containing [3-endorphin (1-27) were observed extending by the whole hypothalamus.

Immunoreactive fibres In the mamillar region (from A 8.0 to A 9.5), a low density of immunoreactive fibres was observed in the corpus mamillare (Fig. 1B) and in the nucleus mamillaris lateralis (Fig. 1B), as well as a moderate density of fibres was located above both nuclei (Fig. 1B). In the periventricular region (from A 11.5 to 13.5), a moderate density of immunoreactive fibres was found in the region located near the ventricle (Fig. 1D, E, Fig. 2C) and a high density in the nucleus arcuatus (Fig. 1D, E, Fig. 2D, E). In the medial region (from A 8.5 to A 14.5), single immunoreactive fibres were found in the nucleus supraopticus (Fig. 1D-F) and in the caudal part of the nucleus suprachiasmaticus (Fig. IF), whereas a low density was located in the caudal part of the hypothalamus posterior (Fig. 1B) and in the region located above the tractus opticus (Fig. 1E, Fig. 3-4). In the nucleus hypothalami ventromedialis (Fig. 1D, Fig. 3B), rostral part of the nucleus suprachiasmaticus, hypothalamus dorsomedialis (Fig. 1E), area


266 Cove~as et al

hypothalamica dorsalis (Fig. 1C, D, Fig. 3C), rostral part of the hypothalamus posterior (Fig. 1C), hypothalamus anterior and in the nucleus ffliformis (Fig. 1D, E) a moderate density of [~-endorphin-ir fibres was observed. Finally, a high density of immunoreactive fibres was found in the nucleus periventricularis hypothalami (Fig. 1E, Fig. 3D). In the lateral hypothalamus (from A 9.0 to A 13.0), a low density of immunoreactive fibres was found in the hypothalamus lateralis (Fig. 1B-E, Fig. 3E). Finally, in the preoptic area (from A 14.0 to A 15.0), a high density of fibres containing [~-endorphin (1-27) was observed in the regio praeoptica (Fig. IF, Fig. 3F).

Tracts Single immunoreactive fibres were observed in the fornix (Fig. 1 C-F).

Immunoreactive cell bodies Perikarya containing [3-endorphin (1-27) were observed in the periventricular and medial regions of the mediobasal hypothalamus. Thus, a high density of such neurons were found in the nucleus arcuatus (Fig. 1D, E, Fig 2D, E) and a low density in the nucleus hypothalami ventromedialis (Fig. 1D).

DISCUSSION

The present report is the first detailed study of the distribution of [~-endorphin-ir fibres and perikarya in the cat diencephalon, using an immunoperoxidase technique. Moreover, we have demonstrated that [~-endorphin-ir structures are widely distributed in the same region of the feline.

Comparison of the distribution of ~-endorphin in the mammalian diencephalon

Biochemical (based on radioimmunoassay in microdis- sected brain nuclei) and immunochemical mapping have been carried out in the CNS of the rat, cat and human in order to know the distribution of [3-endorphin (1-31) ([~- endorphin) or pro-opiomelanocortin. 9,14<¢22 On comparing our resuks with a previous study carried out on the distribution of [~-endorphin immunoreactive structures in the cat hypothalamus, 9 we observed that a similar pat- tern of distribution of both [~-endorphin (1-27) and 13- endorphin appears in some nuclei of the hypothalamus such as arcuatus, hypothalamus dorsomedialis and hypothalamus lateralis, as well as in the periventricular region. However, there are differences since cell bodies containing [~-endorphin (1-27) were observed in the nucleus hypothalami ventromedialis; nevertheless, in this hypothalamic nucleus no [~-endorphin immunoreactive cell body was found in the feline? On the contrary,

Micevych & Elde 9 found cell bodies containing 13-endorphin in the nuclei tuberomamillary, premamfllary and anterior mamillary in which we did not find [~-endorphin-ir perikarya. Moreover, we observed I3-endorphin-ir fibres in the hypothalamic nuclei suprachiasmaticus, supraopticus, periventricularis hypothalami, area hypothalamica dorsalis and regio praeoptica in which Micevych & Elde 9 did not visualize B-endorphin. Thus, it seems that fibres containing ~-endorphin (1-27) and [~- endorphin (1-31) immunoreactive cell bodies are, respectively, more widely distributed in the cat hypothalamus than [3-endorphin (1-31) immunoreactive fibres and [3-endorphin-ir perikarya. At present, we have no data to explain these differences since both peptides are post-translational products of the same precursor, pro- opiomelanocortin, lz,22 However, these discrepancies may be due to: (1) a different kind of processing of [3-endorphin precursor; (2) a complete intraneuronal segregation of [3-endorphin and [3-endorphin (1-27) (see ref. s). Further research would be necessary to discover the ori- gin of such discrepancies.

Our results are, in general, in agreement with the findings described by Gramsch et a116 in the human brain, using radioimmunoassay techniques. These authors observed different levels of ~-endorphin in the medial region of the thalamus, in the corpus geniculatum laterale and in the mamillar, anterior and posterior regions of the hypothalamus. In all these regions, we found [~- endorphin-ir structures in the cat. However, in humans, [3-endorphin was located in the nucleus pulvinar, in which no immunoreactivity was visualized in the feline.

Moreover, the findings observed in this work are in agreement with those found concerning the distribution of [3-endorphin in the rat thalamus and hypothalamus. Thus, in the thalamus, [~-endorphin 14,15 (in the rat) and [~- endorphin (1-27) (in the cat) were visualized in the following thalamic nuclei: medialis dorsalis, habenularis medialis, habenularis lateralis, periventricularis anterior, reuniens, centralis medialis, centralis lateralis, lateralis posterior, ventralis anterior, lateralis dorsalis and zona incerta. However, some differences merit comment. We visualized ~-endorphin-ir structures in the feline, but no [~-endorphin has been found in the rodent 14,1s in the nuclei parataenialis, rhomboidens, parafascicularis, ventralis medialis, geniculatum laterale and geniculatum mediale. By contrast, [~-endorphin was found in the rat 14,~5 in the nuclei paracentralis, ventralis lateralis, ventralis posterolateralis and ventralis posteromedialis, whereas in these nuclei ~-endorphin (1-27) was not observed in the feline. In the hypothalamus, the distribution of [3-endorphin and [3-endorphin (1-27) is identical in both animals, except in the nucleus mamfllaris lateralis, in which [3-endorphin (1-27) was observed in the cat, but no [3-endorphin was found in the rodent24,~5



Fig. 2 JLendorphin-ir cell bodies and fibres in the cat diencephalon. (A) Anteriority 10.5. Immunoreactive fibres (arrows) in the nucleus periventricularis anterior. (B) Anteriority 11.5. High power image of an immunoreactive fibre in the nucleus reuniens. (C) Anteriority 12.0. Immunoreactive fibres ("arrows) in the region located near the ventricle (V). (D) Anteriority 11.5. [3-endorphin-ir fibres (arrows) and perikarya (head arrows) in the nucleus arcuatus. (E) Anteriority 12.5. Immunoreactive fibres (arrows) and cell bodies (head arrows) in the nucleus arcuatus. V: ventricle. Scale bar: 100 #m.

Thus, both peptides were found, for example, in the nuclei arcuatus, hypothalami ventromedialis, supraopticus, suprachiasmaticus, corpus mamillare, periventricularis hypothalami, hypothalamus dorsomedialis, area hypothalamica dorsalis, hypothalamus posterior, regio praeoptica, hypothalamus anterior and hypothalamus lateralis.

Moreover, in general, the distribution of [3-endorphin (1-27) in the cat diencephalon is quite similar to that found on the distribution of pro-opiomelanocortin in the same forebrain region of the rat. 22 Thus, ]3-endorphin (1-27) and pro-opiomelanocortin were found, for example, in the following diencephalic nuclei: medialis dot- sails, parataenialis, habenularis medialis, habenularis lateralis, periventricularis anterior, rhomboidens, reuniens, centralis lateralis, zona incerta, arcuatus, hypothalami ventromedialis, suprachiasmaticus, periventricularis hypothalami, hypothalamus dorsomedialis, area hypothalamica dorsalis, hypothalamus posterior, hypothalamus anterior, hypothalamus lateralis and regio praeoptica.

In sum, it seems that the distribution of [~-endorphin (1-27) in the cat diencephalon is quite similar to that found in the rat diencephalon for [~-endorphin and pro- opiomelanocortin, although in the hypothalamus such distribution is more similar than in the thalamus.

Comparison of the distribution of pro-enkephalin-, pro- dynorphin- and pro-opiomelanocortin-derived peptides in the cat diencephalon

In the literature no study appears on the distribution of pro-dynorphin-derived peptides in the cat diencephalon. However, we have described, in the cat thalamus 1°,11 and hypothalamus/2 the distribution of a pro-enkephalin- derived peptide, the methionine-enkephalin. In general, the location of immunoreactive fibres containing methionine-enkephalin or [3-endorphin (1-27) in the cat diencephalon is quite similar, since in the thalamus both kinds of fibres were observed in the epithalamus (nuclei periventricularis anterior and habenularis) and in the midline region (e.g. nuclei centralis medialis, rhom-


268 Cove#as et al

Fig. 3 Fibres containing 13-endorphin (1-27) in the cat hypothalarnus. (A) Anteriority 12.5. Immunoreactive fibres (arrows) in the region located above the tractus opticus (TO). (B) Anteriority 11.5. Immunoreactive fibres (arrows) in the nucleus hypothalami ventromedialis. Note an immunoreactive cell body (head arrow). (C) Anteriority 10.5. Immunoreactive fibres (arrows) in the area hypothalamica dorsalis. (D) Anteriority 12,0. Immunoreactive fibres (arrows) in the nucleus periventricularis hypothalami. (E) Anteriority 12.5. An immunoreactive fibre (arrows) in the hypothalamus lateralis. (F) Anteriority 14,0. Fibres (arrows) containing ~-endorphin (1-27) in the regio praeoptica. V: ventricle. Scale bar: 100 #m.

boidens, reuniens), as well as in nuclei located near the midline (e.g. nuclei medialis dorsalis and parafascicularis), whereas in the hypothalamns fibres containing [3- endorphin (1-27) or methionine-enkephalin ~2 were visualized in almost all the hypothalamic nuclei (e.g. arcuatus, hypothalami ventromedialis, suprachiasmaticus, area hypothalamica dorsalis, filiformis, hypothala-

mus lateralis, regio praeoptica). The anatomical relationship of both opioid peptides is also supported by physiological studies. 23-25 Thus, it has been demonstrated that ~-endorphin can be released from hypothalamic cells by chemically induced membrane depolarization, suggesting that the peptide could play a role as neuroregulator in the brain? s Moreover, the release of methionine-



enkephalin by [~-endorphin has been demonstrated in both rat and catY ,24 These findings in conjunction with our anatomical data suggest that [~-endorphin (1-27) could regulate the release of methionine-enkephalin in the cat diencephalon.

There is a widespread distribution of cell bodies containing methionine-enkephalin in the cat thalamus and hypothalamus2 °-12 However, [3-endorphin-ir perikarya were only visualized in two hypothalamic nuclei (arcuatus and hypothalami ventromedialis). In these nuclei were also observed perikarya containing methionine- enkephalin.~2 It is obvious on the basis of the morphological features of the two cell populations in the cat, the existence of separate populations of methionine- enkephalin-, and/3-endorphin-ir perikarya, as it has been previously reported in other mammals (for review see ref. 22).

Anatomical relationship of ~-endorphin (1-27) with other neuropeptides in the cat diencephalon

In general, neuropeptides are located in the major brain regions (e.g. the hypothalamus is the richest region in the CNS containing neuropeptides, as well as all neuropeptides have been located in such diencephalic region)) 4 Thus, several neuropeptides can be observed in the same diencephalic nuclei. The distribution of fibres and cell bodies containing vasoactive intestinal polypeptide, substance P, neurokinin A, neuropeptide Y, cholecystokinin, neurotensin and somatostatin has been previously described in the cat diencephalon. 1-8 In general, cell bodies containing the above-mentioned neuropeptides were broadly distributed in the cat diencephalon than those containing ~-endorphin (1-27), whereas on comparing the distribution of ~-endorphin-ir fibres with the location of immunoreactive fibres containing the above-mentioned seven neuropeptides, it seems that the distribution is quite similar. Furthermore, in the cat diencephalon, the anatomical relationship between ~- endorphin-ir fibres and substance P-, neurokinin A-, neuropeptide Y-, neurotensin-, and somatostatin-ir fibres is greater than between ~-endorphin-ir fibres and vasoactive intestinal polypeptide processes.

Thus, all the mentioned neuropeptides, except the vasoactive intestinal polypeptide, have been observed in fibres in the cat diencephalic nuclei habenularis lateralis, periventricularis anterior, centralis medialis, reuniens, rhomboidens, medialis dorsalis, parataenialis, area hypothalamica dorsalis, hypothalami ventromedialis, hypothalamus posterior, hypothalamus anterior, suprachiasmaticus, supraopticus, hypothalamus lateralis and arcuatus. These data suggest a possible interaction among some of the six above-cited neuropeptides in the diencephalon of the cat, and an elaborate modu-

lation of functions in which these diencephalic nuclei are involved. Thus, for example, an interaction between ~-endorphin (1-27) and neuropeptide Y could be possible, since in addition to the close anatomical relationship of both peptides shown in the cat diencephalon, our findings are supported by observations carried out by other authors in the rat. 26,2z Thus, neuropeptide Y has been observed innervating cell bodies containing/3-endorphin in the rat mediobasal hypothalamus, 26 as well as in the nucleus arcuatus of the rodent, it has been described a regulation of pro-opiomelanocortin gene expression by neuropeptide y.2z

In the cat nucleus arcuatus, the existence of cell bodies containing neuropeptides (e.g. substance P, neuropeptide Y, neurotensin) has been described. 2,5,z Mthough, there is an identical location of the immunoreactive perikarya containing such neuropeptides and ~-endorphin-ir cell bodies, a possible coexistence of [3-endorphin (1-27) with some of the three mentioned neuropeptides cannot be suggested, since the morphological characteristics of [3- endorphin-ir perikarya are quite different from those showed by the neuronal populations containing substance P, neuropeptide Y or neurotensin. 2,5,z

Possible J3-endorphin (1-27)-containing pathways in the cat

In the mammalian brain (e.g. rat), ~-endorphin immunoreactive perikarya have been only located in the nucleus tractus solitarius 28,29 and in the arcuate nuclear region (mediobasal hypothalamus) which includes the nuclei arcuatus and hypothalami ventromedialis. 1z28 It is also known that in the rat, axonal fibres from the cell group located in the arcuate nuclear region project to a great number of brain areas including several thalamic and hypothalamic nuclei, lz22'as in which ~-endorphin immunoreactive fibres were also found, lz,22,28 In the feline, we observed [~-endorphin-ir perikarya in the arcuate nuclear region, but not in the nucleus tractus solitarius, in which a low density of fibres containing ~-endorphin (1-27) was only found. However, this observation does not indicate that such brainstem nuclei are devoid of immunoreactive cell bodies, since the lack of such perikarya could be due to the place (lateral ventricle) in which the drug was injected, as this is a region located far from the nucleus tractus solitarius and colchicine would not probably diffuse to such nucleus. Thus, colchicine might be injected in a region located near the cat nucleus tractus solitarius (e.g. the fourth ventricle, where the drug would probably reach the nucleus), in order to know the presence or not of cell bodies containing 13- endorphin (1-27) in such a nucleus.

According to the morphological data observed in the feline, it appears that neurons containing ~-endorphin

© Pearson Professional Ltd 1996 Neuropeptides (1996) 30(I), 261-271

270 Cove5as et al

(1-27) (located in the arcuate nuclear region) have extensive projections throughout the cat thalamus and hypothalamus, as it occurs in the rodentY ,z2'2s Thus, in the cat diencephalic nuclei in which we found immunoreactivity (except the arcuate nuclear region), [3- endorphin-ir fibres were found but no immunoreactive cell body was visualized, suggesting these data that such nuclei receive ~-endorphin (1-27) afferents arising from cell bodies in the arcuate nuclear region. Moreover, B- endorphin-ir fibres also terminate within the arcuate nuclear region, since a high or moderate density of immunoreactive fibres was visualized in this region. However, in order to demonstrate such a hypothesis, the employment of both immunocytochemical and tract- tracing techniques is required, as well as the demonstra- tion of [3-endorphin-ir neurons in the nucleus tractus solitarius, since this nucleus projects to many pro-opiomelanocortin-rich forebrain areas. 2z

Effects of intraventricular administration of colchicine

It has been demonstrated that colchicine has neurotoxic effects on certain CNS neuronsp ° producing the death of selected neuronal populations. It has also been shown that neuropeptide mRNA expression can be induced by the drug, 31 raising the possibility that certain neuropeptides may not be expressed under normal conditions. In our study, the neurotoxicity of colchicine is rejected, since in animals treated with the drug, the morphology of ~-endorphin-ir celi bodies was preserved, as well as sections stained with Nissl's procedure showed that the morphology of the tissue was also preserved. Moreover, the number of immunoreactive cell bodies was greater in treated than in non-treated animals, indicating that colchicine did not induce cellular death. Regarding the above-mentioned second point, 3~ the presence of cell bodies containing J3-endorphin (1-27) in the nucleus arcuatus of control cats suggests that the neuropeptide is expressed under normal conditions. Such expression could be possible by the action of colchicine in the nucleus hypothalami ventromedialis, since [3-endorphin- ir cell bodies were found in treated animals but not in control ones. In the cat, however, this fact is quite improbable, since in the rat a neuronal population containing ~-endorphin has been well established 1~z,28 and we have indicated a similar distribution of [3-endorphin and [3-endorphin (1-27) in the rat and cat diencephalon. Thus, the presence of [~-endorphin-ir perikarya in the nucleus hypothalami ventromedialis is probably due to the disruptive action of colchicine on the microtubule systems and therefore cell body levels of ~-endorphin (1-27) were increased. However, an in situ hybridization study should be carried out in order to find out whether or not [3-endorphin (1-27) neuronal expression in the

nucleus hypothalami ventromedialis is due to the action of colchicine.

Possible physiological functions of [3-endorphin (1-27) in the cat diencephalon

The presence of ~-endorphin (1-27) at multiple sites in the cat diencephalon implies that the peptide serves different functions at these sites.

The anatomical distribution of B-endorphin-ir structures in the midline thalamic region of the cat suggests that the neuropeptide is preferentially concentrated in regions devoted to a motivational or affective aspect of sensory transmission instead of regions (e.g. the specific thalamic nuclei) involved in discriminative functions. 11 Moreover, the presence of B-endorphin-ir fibres in the nucleus medialis dorsalis indicates that [~-endorphin (1-27) might be involved in vigilance and attentive behaviour. 32

In the cat hypothalamus, [3-endorphin (1-27) could be involved, for example, in feeding and affective defense behaviour and in termogenesis regulat ion# ,~4 since immunoreactive structures were found in the nucleus hypothalami ventromedialis. Moreover, the [~-endorphin (1-27) immunoreactivity observed in the nucleus suprachiasmaticus could indicate a possible role in the control of circadian rhythms and/or in visual processes# and the presence of [3-endorphin-ir fibres in the nucleus periventricularis hypothalami suggests the involvement of the peptide in stress mechanismsY Although furore works are required to elucidate the physiological signifi- cance of [~-endorphin (1-27) in the cat diencephalon, we hope that our anatomical study will assist investigators in order to know the diverse actions of such peptide in the cat thalamus and hypothalamus.

ACKNOWLEDGEMENTS

The authors wish to thank to Mr Nicholas Skinner for stylistic revision of the English text. This work has been supported in part by the Junta de Castilla y Le6n (SA64/93), Spain and the DGICYT (PB93/0992 and PB91/0769), Spain.

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