Brain Research Bulletin. Vol. 31, pp. 279-285, 1993 0361-9230/93 $6.00 + .OO Printed in the USA. All rights reserved. Copyright 0 1993 Pergamon Press Ltd.

Distribution of Neurokinin A in the Cat Diencephalon: An Immunocytochemical Study

A. VELASCO,* M. DE LEeN,*, R. COVE&AS, *’ P. MARCO&* J. A. NARVxkEZ,t G. TRAMU,$ J. A. AGUIRREt AND S. GONZALEZ-BAReN?

*Universidad de Salamanca, Facultad de Medicina, Dpto. Biologia Celular y Patologia, Avda. Camp0 Charro s/n, 37007~Salamanca, SpainfUniversidad de MLtlaga, Facultad de Medicina, Dpto. Fisiologia, Ma’laga, Spain

$Laboratoire de Neurocytochimie Fonctionelle, CNRS, URA 339, Universitt de Bordeaux I, France

Received 3 1 July 1992; Accepted 6 October 1992

VELASCO, A, M. DE LE6N, R. COVERAS, P. MARCO& J. A. NARVAEZ, G. TRAMU, J. A. AGUIRRE AND. S. GON- ZALEZ-BARON. Distribution of neurokinin A in the cat diencephalon: An immunocytochemical study. BRAIN RES BULL 31(3/4) 279-285, 1993.-The distribution of neurokinin A-like immunoreactive cell bodies and fibers in the diencephalon of the cat was studied using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive neurons was observed in the nuclei habenularis lateralis, medialis dorsahs, parafaxicularis, hypothalamus posterior, area hypothalamica dorsalis, hypothalamus lateralis, periventricularis hypothalami, above the corpus mamillare, and in the perifornical area, whereas scarce immunoreactive perikarya were visualized in the nuclei reuniens, hypothalami ventromedialis, hypothalamus dorsomediahs, and mamillaris lateralis. The highest density of fibers containing neurokinin A was found in the nuclei periventricularis anterior, rhomboidens, centralis medialis, periventricularis hypothalami, and supraopticus. In the regio praeoptica, area hypothalamica dorsalis, hypothalamus posterior, and in the perifornical area a moderate density of immunoreactive fibers was observed, whereas the nuclei habenularis lateralis, medialis dorsalis, mamillaris lateralis, parataenialis, reuniens, habenularis medialis, tiliformis, hypothalamus dorsomedialis, hypothalami ventromedialis, arcuatus, and suprachiasmaticus showed a low density of neurokinin A immunoreactive fibers.

Neurokinin A Diencephalon Immunocytochemistry Cat

NEUROKININ A (NKA) is a member of the family of the mammalian tachykinin peptides, as well as substance P (SP) and neurokinin B (NKB) (22). These tachykinins have a common C-terminal amino acid sequence and they have been reported to be involved in several physiological roles such as the regulation of smooth muscle contraction, salivation, depolarization of cen- tral neurons, central pressor action, hyperactivity, as well as in- teracts with dopaminergic A- 10 neurons mediating behavioral activation (3,12,24,26). In addition, NKA and SP may be neu- rotransmitters of the baroreceptor reflex in the nucleus tractus solitarii of the rat, and NKB may be a neuromodulator on car- diovascular responses in the same nucleus (25). Finally, NKA and NKB may have a neurotransmitter/neuromodulator role in the cat substantia nigra (10). It is also known that SP and NKA are derived from the preprotachykinin A gene, whereas NKB is derived from the preprotachykinin B gene.

In general, the distribution of tachykinins in the mammalian CNS has been characterized using immunocytochemistry and radioimmunoassay techniques (1,2,8,11,13,18,2 1,23,27,29-3 1). However, these studies have been carried out on the distribution of SP in the CNS of the rat, cat, monkey, and humans, whereas, for example, the anatomical localization of NKA and NKB has

been carried out mainly in rats. In the cat, the data available on the NKA show that the peptide enhances the depression of spinal nociceptive neurons caused by cutaneously applied vibration and that NKA is released in the spinal cord following injection of a knee joint with kaolin and carrageenan ( 16).

In the cat diencephalon, we have studied the anatomical dis- tribution of the neuropeptides methionine-enkephalin, SP, neu- ropeptide Y, somatostatin-28 ( 1- 12) and neurotensin (2,4- 7,19,20), but no data are available on the distribution of fibers and cell bodies containing NKA in this region of the feline brain. Thus, in the present work we attempted to study the distribution of NKA in the cat diencephalon using an immunoperoxidase technique and to compare our findings with the distribution of the above-mentioned neuropeptides previously describe in the feline diencephalon.

METHOD

Eight male adult cats (2-3 kg) were used. Three animals, under deep ketamine anesthesia (40-50 mg/kg), received uni- lateral intraventricular injections of colchicine in the lateral ventricle (300 pg in 5 ~1 of distilled water). The other five cats

’ To whom requests for reprints should be addressed.

279

280 VELASCO ET AL.

were not treated with the drug. Two days after the injection, the treated animals as well as the untreated ones were anesthetized with ketamine and perfused via the ascending aorta with 4 1 of 4% ~rafo~aldehyde in 0.15 M phosphate buffer (PB) (pH 7.2). The brains were removed, the diencephalon dissected out and postfixed in the same solution for 12 h. With a Vibratome, 60 pm frontal sections were cut and processed for immunostaining. The sections were incubated in PB containing 1% normal sheep serum and 0.3% Triton X-100 for 30 min. The sections were then placed overnight in the same PB solution containing NKA antiserum diluted l/l~. After a 30 min wash with PB, the sections were incubated for 60 min with sheep antirabbit IgG coupled to horseradish peroxidase as the second antibody, diluted l/250 in PB. Finally, the sections were washed in PB and the peroxidase was revealed by the 33’ diaminobenzidine method. The antiserum used in the present work was purchased from Bachem (Swi~erland). It was raised in rabbits against immu- nogens prepared by coupling the peptide to a carrier protein (human serum albumin) with giutaraldehyde. The specificity of the immunostaining was controlled by the preabsortion of the primary antiserum with synthetic NKA antibody and by omitting the NKA antibody in the first incubation bath. No residual im- munoreactivity was found in either case. Moreover, possible in- terference by endogenous peroxidase was ruled out by staining some sections beginning with the diaminobenzidine step. No reaction was visualized. Immunohistochemical crossreactivities were performed by incubating NKA antiserum with an excess of SP, NKB, eledoisin, or kassinin. In no case was any significant reduction in immunoreactivity observed. Mapping was carried out according to the stereotaxic atlas of Jasper and Ajmone- Marsan ( 17). The same atlas was used for the terminology of the diencephalic nuclei. Finally, the term neurokinin A-like im- munoreactive (NKA-ir) was used to described the staining results in our material.

RESULTS

Figure 1 shows the dist~bution of NKA-ir fibers and cell bodies in the diencephalon of the cat. The hypothalamus showed a higher density and a more widespread distribution of NKA-ir structures than the thalamus. Thus, in the thalamus, NKA-ir perikarya were found in the nucleus (n.) habenularis lateralis, n. medialis dorsalis, n. parafascicularis and in the n. reuniens, whereas in the hypothalamus, immunorea~ive neurons were visualized, e.g., in the n. mamillaris lateralis, n. hypothalami ventromedialis, n. periventricularis hypothalami, area hypoth- alamica dorsalis, hypothalamus posterior, hypothalamus dor- somedialis, above the corpus mamillare, and in the perifomical area. Moreover, NKA-ir fibers were mainly located in the thal- amus in the midline nuclei or in nuclei located near the midline, being absent in the lateral thalamic regions, whereas in the hy- pothalamus, immunoreactive fibers were found in almost all the nuclei.

At anteriority (A) 5.0 and A 7.0 (not shown in Fig. I), re- spectively, was found a very low density of NKA-ir fibers in the dorsal and lateral parts of the corpus geniculatum mediale and a moderate densitv of both immunoreactive fibers and cell bodies in the n. pamfa~~cula~s (Fig. 2A).

At A 7.5 (Fig. 1 A), a high density of NKA-ir cell bodies was observed in the n. habenularis lateralis (Fig. 2B) and a moderate density in the n. medialis dorsalis (below the n. habenularis la- teralis), below the n. subparafascicularis, and between this latter nucleus and the ventricle. A scarce number of NKA-ir fibers was observed in the stria medullaris, n. habenularis medialis, n. ha~nuia~s lateralis, and n. medialis dorsalis, whereas a high

density of immunoreactive fibers was visualized in the n. peri- ventricularis anterior. In addition, NKA-ir fibers were also lo- cated ventrally along the midline (moderate density) and ex- tending towards the substantia nigra. In this, a large number of NKA-ir processes was observed.

At A 8.5 (Fig. IB) NKA-ir cell bodies were observed above the corpus mamillare (Figs. 2C, D) and in the n. mamillaris lateralis. In these regions a high and a low density, respectively, of NKA-ir cell bodies was visualized. Moreover, a large number of immunoreactive cell bodies was visualized above the com- missura supramamilla~s. Scarce immunoreactive fibers were observed extending from the corpus mamillare towards the pe- dunculus cerebralis. Dorsally, a few immunoreactive fibers were found in the region of the n. lateralis dorsalis nearest to the midline, in the n. habenularis medialis, in the stria meduliaris, in the n. habenularis lateralis, as well as in the midline and dorsal regions of the n. medialis dorsalis, and in the same nucleus in the region placed below the n. habenularis lateralis. Finally, a high density of NKA-ir fibers was found in the n. centralis medialis and above the corpus mamillare and a low density in the n. interventricularis and in the n. mamillaris lateralis and in the midline region extending from this latter nucleus to the commissura supramamiiaris.

At A ii .O (Fig. I CT), a low density of NKA-ir cell bodies was found in the thalamus in the n. reuniens. In the hypothalamus (Fig. 3A), a high density of NKA-ir cell bodies was observed in the hypothalamus posterior, the perifornical area (Fig. 2E), and in the region extending ventrally from this area to the n. hy- pothalami ventromedialis. A moderate density of immunoreac- tive cell bodies was also found in the area hypothalamica dorsalis (Fig. 2F) and in the hy~thalamus lateralis (Fig. 3B), whereas in the n. hypothalami ventromedialis, a few NKA-ir cell bodies were visualized. In the thalamus, immunoreactive fibers were found in the midline region. Thus, a low density of immuno- reactive fibers was located in the stria medullaris, n. parataenialis, and n. reuniens, whereas a high density was visualized in the n. periventricuiaris anterior, n. rhomboidens, and in the n. centralis medialis. In the hy~thalamus. scarce immunoreactive processes were found in the n. filiformis and in the n. hypothalami ven- tromedialis. The perifornical area, the hypothalamus lateralis. area hypothalamica dorsalis, and the hypothalamus posterior showed a moderate density of NKA-ir fibers.

At A 12.5 (Fig. 1 D), a low density of NKA-ir cell bodies was found in the n. reuniens. A high density of immunoreactive perikarya was visualized below the h~othalamus dorsomedialis and close to the ventricle, a moderate density in the hypothal- amus lateralis and in the n. periventricularis hypothalami, and a low density in the hypothalamus dorsomedialis. At the same level, scarce immunoreactive fibers were visualized in the stria medullaris, n. parataenialis, n. reuniens, n. fiiiformis, hypo- thalamus dorsomedialis, and n. arcuatus, whereas a high density of NKA-ir fibers was found in the n. ~~vent~cula~s anterior (Fig. 3C), n. periventt-icularis hypothalami, and n. supraopticus. In addition, in the hypothalamus lateralis, in the region situated near the ventricle and below the hypothalamus dorsomedialis, as well as in the regions surrounding the n. filiformis and the fornix and in the regions extending from the n. supraopticus to the midline and from the same nucleus to the cap&a intema, a moderate density of NKA-ir fibers was found.

At A 13.5 (Fig. 1 E) a high density of NKA-ir neurons was found near the ventricle between (Figs. 3D, E) the n. peri- ventricularis hypothalami, and the n. suprachiasmaticus, and a moderate density in the n. periventricularis hypothalami. In addition, at this level two clusters of immunoreactive cell bodies (moderate density) were found laterally. A high density

NKA IN THE CAT DIENCEPHALON 281

A A7.5

FIG. 1. Distribution of NRA-ir fibers and cell bodies in frontal planes of the diencephalon of the cat corresponding to the posteroanterior ste- reotaxic plane levels A 7.5 to A 13.5 of the Jasper and Ajmone-Marsan (17) stereotaxic atlas. Immunoreactive fibers are represented by contin- uous lines, whereas cell bodies are indicated by closed circles, their shape being related to the density of perikarya. (large filled circle, high density: > IO cell bodies; medium filled circle, middle density: S-IO cell bodies; small filled circle, low density: <5 cell bodies). The anterior&y (A), in mm with respect to the zero stereotaxic point of each section is indicated at the lower right. AHD, area hypothalamica dorsalis; AM, n. anterior medialis; ARC, n. arcuatus; AV, n. anterior ventralis; CH, chiasma op- ticum; CI, capsula intema; CL, n. centralis lateralis; CM, n. centrum medianum; FIL, n. filiformis; FX, fomix; GL, corpus geniculatum lat- erale; GM, corpus geniculatum mediale; HA, hypothalamus anterior; HBL, N. habenularis lateralis; HBM, n. habenularis medialis; HDM, hypothalamus dorsomedialis; HL, hypothalamus lateralis; HP, hypo- thalamus posterior; IV, n. interventricularis; LD, n. lateralis dorsalis; LP, n. lateralis posterior, MD, n. medialis dorsalis; ML, n. mamillaris lateralis; MM, corpus mamillare; NCM, n. centralis medialis; NHVM, n. hy- pothalami ventromedialis; PED, pedunculus cerebralis; PUL, n. pulvinar; PVA, n. periventricularis anterior; PVH, n. periventricularis hypothalami; R, n. reticularis; RE, n. reuniens; RH, n. rhomboidens; S, stria medullaris; SCH, n. suprachiasmaticus; SMX, commissura supramamillaris; SN, substantia nigra; SO, n. supraopticus; VA, n. ventralis anterior; VL, n. ventralis lateralis; VM, n. ventralis medialis; VPL, n. ventralis postero- lateralis; VPM, n. ventralis postero-medialis.

VELASCO ET AL.

FIG. 2. NKA immunoreactivity in the diencephalon of the cat. (A) Anteriority 7.0. Immunoreactive fibers (small arrows) and cell bodies (large arrows) in the n. parafascicularis. V: ventricle (X85). (B) Anteriority 7.5. NKA-ir cell bodies (large arrows) and fibers (small arrows) in the n. habenularis lateralis (X85). (C) Anteriority 8.5. Clusters of immunoreactive neurons above the corpus mamillare. Note NKA-ir perikarya (arrows) located medially. HP: h~thalamus posterior: MM: corpus mamiliare; SMX: commissura supramamiila~s; V: ventricle (X 17). (D) Anteriority 8.5. High power image of the delimited area in C (X85). (E) Anteriority 11.0. NKA-ir cell bodies and fibers (arrows) near the fomix (FX) (X85). (F) Anteriority 10.0. Fibers and cell bodies (arrows) containing NKA located in the area hypothalamica dorsalis. TMT: tractus mamillo-thalamicus; V: ventricle (X34).

NKA IN THE CAT DIENCEPHALON 283

FIG. 3. NKA-ir fibers and cell bodies in the cat thalamus and hypothalamus. (A) Anteriority 11.0. A low magnification of the hypothaiamus. Immunoreactive cell bodies (arrows) showing a widespread distribution. AHD: area hypothalamica dorsalis; F’Xz fomix; HP: hypothalamus posterior; NHVM: n. hy~thalami ventrom~ialis; V: ventricle (X34). (B) Anterior&y 11.0. Immunomacti~e fibers and cell bodies (arrows) located in the hypothalamus lateralis. CI: cap&a intema; FX: fomix (X34). (C) Anteriority 12.5. NKA-ir fibers (arrows) in the n. periventricularis anterior (X85). (D) Anteriority 13.5 . Clusters of NKA-ir cell bodies located near the ventricle (V) (X34). (E) Anteriority 13.5. High power image of the area delimits in D (X85).

of NKA-ir fibers was found in the n. periventricularis an- terior, n. periventricularis hypothalami, and n. supraopticus, whereas a low density of immunoreactive fibers was ob- served in the n. suprachiasmaticus and in the hypothalamus anterior. Moreover, a moderate density of NKA-ir fibers was

visualized above the chiasma opticum, along all the midline and surrounding the n. periventricularis hypothalami and the fornix. Finally, in the region praeoptica (not shown in Fig. 1) a moderate density of immunoreactive fibers was found.

284 VELASCO ET AL.

DISCUSSION

The present work is the first report that shows in detail the distribution of NRA-ir structures in the mammalian dienceph- alon, using an indirect immunoperoxidase technique.

However, future studies are needed in order to corroborate this hypothesis.

In comparison with a study on the cellular localization of SP and NRA-encoding preprotachykinin (PPT) mRNA in the fe- male brain (13), our results are in agreement in some features. Thus, Harlam et al. (13) observed, in the rat, PPT neurons in the n. hypothalami ventromediahs, hypothalamus dorsomedialis, the perifomical area, and above the corpus mamillare, in which we found NRA-ir cell bodies in the feline. Moreover, these au- thors observed neurons containing PPT in the n. arcuatus but no PPT cell body in the n. periventricularis hypothalami. How- ever, we have visualized in the cat immunoreactive cell bodies in the latter nucleus but none in the former. Moreover, Harlam et al. (13) did not find labeled neurons containing PPT in the rat thalamus; however, we found NRA-ir perikarya in the cat in the n. habenularis lateralis, n. medialis dorsalis, and n. reu- niens. These observations reveal that there are some differences on the distribution of neurons expressing the PPT gene in the rat diencephalon in comparison with the distribution of NKA- ir cell bodies observed in the same region in the cat. These dis- crepancies could be due to technical considerations and/or spe- cies differences.

In addition, our results are in agreement with other studies in which radioimmunoassay techniques has been used. In this sense, using these techniques NRA has been described in the thalamus and hypothalamus of both rat and golden hamster (9,29), as we found using immunocytochemical techniques in the cat diencephalon. In addition, more NRA was found in the rat hypothalamus than in the thalamus (28). In the cat, we have also observed more immunoreactive structures in the hypo- thalamus than in the thalamus.

In addition, an anatomical relationship between NRA and neurotensin, somatostatin, or methionine-enkephahn can be pointed out, because, for example, the four peptides have been found in the n. habenularis lateralis, n. medialis dorsalis, n. per- iventricularis anterior, n. rhomboidens, n. centralis medialis, n. reuniens, area hypothalamica dorsalis, and hypothalamus pos- terior (4,6,19,20). Finally, the mentioned neuropeptides and neuropeptide Y were found in the n. rhomboidens, n. centralis medialis, n. reuniens, n. medialis dorsalis, and n. periventricularis anterior (4,5,19,20). These data also suggest a possible colocal- ization of such neuroactive substances in the cat diencephalon, as well as a possible interaction among some of the above-men- tioned neuropeptides and an elaborate modulation of functions in which these diencephahc nuclei are involved. The distribution of NRA-ir structures in the diencephalon of the cat suggest that the peptide could be involved in several physiological functions. Thus, for example, the presence of NRA-ir fibers in both n. periventricularis hypothalami and n. supraopticus suggests that NRA might regulate neuronal activity in both nuclei. Moreover, the localization of NRA in the n. suprachiasmaticus could in- dicate a possible role in the control of circadian rhythms and/ or in visual processes, whereas the presence of immunoreactive fibers in the midline thalamic region could be related with mo- tivational or affective aspects of the sensory transmission. In sum, the physiological significance of NRA in the cat dienceph- alon remains to be elucidated.

The distribution of fibers and cell bodies containing methi- onine-enkephalin, neurotensin, somatostatin, SP, and neuro- peptide Y has been described in the cat diencephalon (2,4- 7,19,20). An anatomical relationship between NRA and SP can be suggested for the diencephalon ofthe cat. Thus, both peptides have been found, for example, in the following nuclei: haben- ularis lateralis, periventricularis anterior, parataeniahs, rhom- boidens, centralis medialis, reuniens, area hypothalamica dor- salis, hypothalamus posterior, perifomical area, filiformis, suprachiasmaticus, and supraopticus. This finding is consistent with the results observed by Helke et al. (14) on the distribution of NRA and SP in the spinal cord, because in this region these authors found the same distribution patterns of both peptides. Our data also suggest a possible colocalization of NRA and SP in the cat diencephalon, as has been demonstrated in sensory neurons and in neurons of the medullary raphe nuclei ( 14,lS).

Until now, we have no data indicating whether the NRA-ir perikarya observed in the hypothalamus of the cat are local or projecting neurons. However, according to the morphological data obtained in the cat, it appears that the NRA-ir neurons found, for example, in the n. habenularis lateralis send distant NRA-ir projections, because a high density of NRA-ir perikarya was found in this region as well as a low density of immuno- reactive fibers, whereas the neurons containing NRA located in the area hypothalamica dorsalis, hypothalamus lateralis, and in the n. parafascicularis could be interneurons, because a moderate density of both immunoreactive cell bodies and fibers was ob- served in these regions. Alternatively, these neurons may send distant NRA projections, whereas NRA-ir fibers may be NRA afferents. Finally, it also appears that the n. periventricularis anterior, n. rhomboidens, n. centralis medialis, n. supraopticus, and the regio praeoptica could recive NRA-ir afferents because in all these nuclei a high or moderate density of NRA-ir fibers was observed but no NRA-ir cell body. In sum, the origin of the NRA-ir fibers and knowledge of whether the immunoreactive neurons found in the cat diencephalon are local or projecting neurons should be investigated in future research.

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