Journal of Chemical Neuroanatomy 42 (2011) 89–98

Mapping of somatostatin-28 (1–12) in the alpaca diencephalon

R. Covenas a,*, A. Mangas a,b, L.E. Medina a, M.L. Sanchez a, L.A. Aguilar c, Z. Dıaz-Cabiale d, J.A. Narvaez d

a Institute of Neurosciences of Castilla y Leon (INCYL), Laboratory of Neuroanatomy of the Peptidergic Systems, Salamanca, Spainb University of Salamanca, School of Medicine, Department of Physiology and Pharmacology, Salamanca, Spainc Cayetano Heredia Peruvian University, School of Medicine ‘‘Alberto Hurtado’’, Lima, Perud University of Malaga, School of Medicine, Department of Physiology and Pharmacology, Malaga, Spain

A R T I C L E I N F O

Article history:

Received 23 February 2011

Received in revised form 31 May 2011

Accepted 17 June 2011

Available online 24 June 2011

Keywords:

Somatostatin

Thalamus

Hypothalamus

Camelids

Immunocytochemistry

Lama pacos

A B S T R A C T

Using an immunocytochemical technique, we report for the first time the distribution of

immunoreactive cell bodies and fibers containing somatostatin-28 (1–12) in the alpaca diencephalon.

Somatostatin-28 (1–12)-immunoreactive cell bodies were only observed in the hypothalamus (lateral

hypothalamic area, arcuate nucleus and ventromedial hypothalamic nucleus). However, immunoreac-

tive fibers were widely distributed throughout the thalamus and hypothalamus. A high density of such

fibers was observed in the central medial thalamic nucleus, laterodorsal thalamic nucleus, lateral

habenular nucleus, mediodorsal thalamic nucleus, paraventricular thalamic nucleus, reuniens thalamic

nucleus, rhomboid thalamic nucleus, subparafascicular thalamic nucleus, anterior hypothalamic area,

arcuate nucleus, dorsal hypothalamic area, around the fornix, lateral hypothalamic area, lateral

mammilary nucleus, posterior hypothalamic nucleus, paraventricular hypothalamic nucleus, supra-

chiasmatic nucleus, supraoptic hypothalamic nucleus, and in the ventromedial hypothalamic nucleus.

The widespread distribution of somatostatin-28 (1–12) in the thalamus and hypothalamus of the alpaca

suggests that the neuropeptide could be involved in many physiological actions.

� 2011 Elsevier B.V. All rights reserved.

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Journal of Chemical Neuroanatomy

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1. Introduction

Somatostatin has been implicated in numerous physiologicaleffects. After central administration of this peptide, behaviouralchanges such as difficulty in breathing, excessive grooming,decreased sleep, and hypersensitivity to tactile stimuli have beenreported (Reichlin, 1983). Moreover, somatostatin inhibits therelease of noradrenalin and growth hormone, stimulates the releaseof acetylcholine and serotonin, and exerts an important neural effecton the motor cortex, hippocampus and limbic system (Reichlin,1983). With immunocytochemical and radioimmunoassay techni-ques, three prosomatostatin-derived peptides (somatostatin-28,

Abbreviations: III, third ventricle; AD, anterodorsal thalamic nucleus; AHy, anterior hy

anteroventral thalamic nucleus; ci, capsula interna; CL, centrolateral thalamic nucleus; C

area; Fx, fornix; LD, laterodorsal thalamic nucleus; LG, lateral geniculate nucleus; LH,

nucleus; LP, lateroposterior thalamic nucleus; MD, mediodorsal thalamic nucleus; ME, m

MM, medial mammilary nucleus; opt, optic tract; ox, optic chiasm; PC, paracentral th

nucleus; Po, posterior thalamic nucleus; PV, paraventricular thalamic nucleus; PVH, pa

thalamic nucleus; Rt, reticular thalamic nucleus; s, stria medullaris; SCh, suprachiasma

nucleus; STh, subthalamic nucleus; V, ventricle; VA, ventroanterior thalamic nucleus

ventromedial hypothalamic nucleus; VPL, ventroposterior thalamic nucleus, lateral par

* Corresponding author at: University of Salamanca, Institute of Neurosciences of Cas

Spain. Tel.: +34 923294400x1856; fax: +34 923294549.

E-mail address: covenas@usal.es (R. Covenas).

0891-0618/$ – see front matter � 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.jchemneu.2011.06.006

somatostatin-28 (1–12) and somatostatin-14) have been describedin the central nervous system of rats (Barden et al., 1981; Bennet-Clarke et al., 1980; Benoit et al., 1982; Finley et al., 1981; Guy et al.,1985; Johansson et al., 1984; Palkovits, 1988; Vincent et al., 1985),guinea-pigs (Hokfelt et al., 1974; Tramu et al., 1981), cats (de Leonet al., 1991, 1992; Graybiel and Elde, 1983; Martın et al., 2003),monkeys (Macaca fascicularis, Saimiri sciureus) (Amaral et al., 1989;Campbell et al., 1987; Hendry et al., 1984; Lewis et al., 1986) andhumans (Bennet-Clarke and Joseph, 1986; Bouras et al., 1987;Cooper et al., 1981; Hornung et al., 1992; Sorensen, 1982).Somatostatin-14 is a tetradecapeptide originally isolated from thehypothalamus, somatostatin-28 consists of the entire sequence of

pothalamic area; AM, anteromedial thalamic nucleus; Arc, arcuate nucleus; AV,

M, central medial thalamic nucleus; cp, cerebral peduncle; DA, dorsal hypothalamic

lateral hypothalamic area; LHb, lateral habenular nucleus; LM, lateral mammilary

edian eminence; MG, medial geniculate nucleus; MHb, medial habenular nucleus;

alamic nucleus; PF, parafascicular thalamic nucleus; PH, posterior hypothalamic

raventricular hypothalamic nucleus; Re, reuniens thalamic nucleus; Rh, rhomboid

tic nucleus; SO, supraoptic hypothalamic nucleus; SPF, subparafascicular thalamic

; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VMH,

t; VPM, ventroposterior thalamic nucleus, medial part; ZI, zona incerta.

tilla y Leon (INCYL), Laboratory 14, c/Pintor Fernando Gallego, 1, 37007 Salamanca,

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–9890

somatostatin-14 at its carboxyl terminal, followed by a double pairof basic amino acids and then somatostatin-28 (1–12); the peptidecorresponding to the first 12 amino acids of somatostatin-28 isnamed somatostatin-28 (1–12) (see Benoit et al., 1985).

In general, previous research carried out on South Americacamelids has mainly focused on reproductive mechanisms (Bravoet al., 1996; Correa et al., 1997; Ratto et al., 2005, 2006). No study hasbeen carried out using immunocytochemical techniques on thedistribution of neuropeptides in the central nervous system ofcamelids, except for two recent studies carried out on the distributionof immunoreactive fibers and cell bodies containing leucine-enkephalin or calcitonin gene-related peptide in the alpaca brainstem(deSouza et al., 2007, 2008) and another focused on the colocalizationof calcitonin gene-related peptide and tyrosine hydroxylase in thealpaca brainstem (Marcos et al., 2011). In this sense, no studyaddressing the presence of somatostatin-immunoreactive structuresin the alpaca diencephalon has been reported. It is known that thethalamus and the hypothalamus are diencephalic regions involved inmany physiological actions. Thus, the thalamus receives somato-sensorial, nociceptive, visual, auditive, vestibular, taste and olfactoryinputs (see Covenas et al., 2001), whereas the hypothalamus isinvolved in drinking, food intake, thermoregulation, neuroendocrinecontrol, immunoregulation, circadian rhythms, blood pressure,stress, reproduction and aggressive behaviour (see Swaab, 1997).Accordingly, our aim in this work was to study the distribution ofimmunoreactive fibers and cell bodies containing somatostatin-28(1–12) in both important regions of the central nervous of the alpaca,using an immunocytochemical technique. A further aim was tocompare our results with the distribution of the pre-prosomatosta-tin-derived peptides (e.g., somatostatin-14) previously described inthe mammalian diencephalon. The neuroanatomical findingsreported here will serve in the future to demonstrate the possibleinvolvement of somatostatin-28 (1–12) in the physiological actionsmentioned above in the alpaca diencephalon.

2. Materials and methods

2.1. Animals

The experimental design, protocols, and procedures of this work were performed

under the principles of laboratory animal care and under the guidelines of the ethics

and legal recommendations of Peruvian and Spanish legislation. This work was also

approved by the research commission of the Cayetano Heredia Peruvian University

(Lima, Peru). We used six male adult alpacas (Lama pacos) (70–80 kg) obtained from

the Cayetano Heredia Peruvian University (Faculty of Veterinary Medicine and Animal

Sciences, Lima, Peru). From birth to perfusion, the animals were maintained at 0 m

(sea level) under standard conditions of light and temperature and had free access to

food and water.

2.2. Tissue preparation and immunocytochemistry

The tissue preparation and immunocytochemical procedures used have been

published previously (see de Souza et al., 2008; Marcos et al., 2011). In brief, the

alpacas were deeply anaesthetized with ketamine (10 mg/kg) and xylazine (4 mg/

kg) and perfused via the carotid artery with 3 l of cold 0.9% NaCl and 5 l of cold 4%

paraformaldehyde in 0.15 M phosphate-buffered saline (PBS) (pH 7.2). Dience-

phalons were cryoprotected, and 50-mm frontal sections were cut with a cryostat

and collected in PBS. In general, six out of seven sections were used for

immunocytochemistry (the remaining section was stained with the Nissl

technique). Then, the sections were washed in PBS and pre-incubated for 30 min

in PBS containing 1% normal horse serum and 0.3% Triton X-100 and then incubated

overnight in PBS containing anti-somatostatin-28 (1–12), diluted 1/5000. After

washing in PBS, the sections were incubated for 1 h with biotinylated anti-rabbit

IgG diluted 1/200 in PBS, after which they were washed extensively. The sections

were incubated with Vectastain ABC reagent (diluted 1/100) for 1 h at room

temperature and then rinsed in PBS and Tris–HCl buffer (pH 7.6). The tissue-bound

peroxidase was developed with H2O2, using 3,30-diaminobenzidine as chromogen.

Finally, the sections were rinsed with PBS and coverslipped with PBS/glycerol (1/1).

2.3. Specificity of the antisera

The first antiserum was raised in rabbits against immunogens prepared by

coupling the peptide (synthetic somatostatin-28 (1–12)) to a carrier protein

(human serum albumin) with glutaraldehyde, as previously reported (de Leon et al.,

1991, 1992; Martın et al., 2003). This antibody was obtained at the laboratory of

Professor Gerard Tramu, University of Bordeaux I (France) and was preabsorbed

with the carrier protein and the coupling agent in order to prevent non-specific

immunoreactivity due to the anti-carrier antibodies. This preabsorption was carried

out before the immunocytochemical applications.

In addition, as previously reported (de Leon et al., 1991, 1992; Martın et al., 2003)

here the specificity of the immunostaining was checked by: (1) preabsorption of the

primary antiserum with synthetic somatostatin-28 (1–12) (100 mg per ml of

diluted antiserum) (Fig. 2D); (2) omitting somatostatin-28 (1–12) antiserum in the

first incubation bath (Fig. 2E); and (3) preabsorption of the primary antiserum with

an excess of synthetic somatostatin-28, somatostatin-14, neuropeptide Y,

angiotensin II, substance P, cholecystokinin and methionine-enkephalin. In all

cases, the results found confirmed the specificity of the antisera used in this study.

Moreover, in order to avoid possible interference by endogenous peroxidases, free-

floating sections were treated with a mixture of NH3, NaOH and H2O2 before to

carrying the immunocytochemical procedure (Guntern et al., 1989).

2.4. Mapping

Contiguous sections to that reacted for somatostatin-28 (1–12) were stained for

Nissl substance with cresyl violet. Moreover, some sections were counterstained

with haematoxylin–eosin once the immunocytochemical technique had been

carried out. The different areas of the diencephalon were identified with the aid of

brain atlases for Lama glama (available from the Mammalian Brain Collections of the

University of Wisconsin, Madison, U.S.A.) and non-camelid mammals (Jasper and

Ajmone-Marsan, 1966; Paxinos and Watson, 1998). For the nomenclature of the

alpaca diencephalic nuclei, the atlases of Paxinos and Watson (1998) and Jasper and

Ajmone-Marsan (1966) were used.

The density of the immunoreactive fibers was established according to an

already described protocol (Covenas et al., 2004; de Souza et al., 2008) as high,

moderate, low and single (Table 1). This involved viewing the sections under

illumination by light at constant magnification with reference to photographs of a

defined series of densities (high, moderate, low) established previously. As

previously described (see Belda et al., 2000), immunoreactive fibers were measured

using a micrometer grid, being considered short (<90 mm), medium (90–120 mm)

or long in length (>120 mm). In addition, a high density of immunoreactive cell

bodies was considered when more than 20 cell bodies/region/section were found;

moderate when 10–20 cell bodies/region/section were present, and low when

fewer than 10 cell bodies/region/section were observed (see Belda et al., 2000). Cell

bodies were considered small when the diameter observed was below 15 mm;

medium-sized when it was between 15 and 25 mm; and large when it was above

25 mm (see Belda et al., 2000). The sizes of the immunopositive cell bodies were

measured using a micrometer grid when the nuclei were in the focal plane.

Photomicrographs were obtained with an Olympus DP50 digital camera

attached to a Kyowa Unilux 12 microscope. To improve the visualization of the

results, only the brightness and contrast of the images were adjusted, with no any

further manipulation of the photographs. Adobe Photograph 6.0 software was used

to view the images and adjust their brightness and contrast.

3. Results

Table 1 shows the density of the immunoreactive structurescontaining somatostatin-28 (1–12) in the alpaca diencephalon,whereas Fig. 1 shows the widespread distribution of thesestructures from rostral (Fig. 1A) to caudal (Fig. 1E) diencephalicregions. In the alpaca diencephalon, the presence of immunoreac-tive cell bodies was quite restricted, since cell bodies containingsomatostatin-28 (1–12) were only observed in three hypothalamicnuclei: a high density of immunoreactive cell bodies was observedin the lateral hypothalamic area (Figs. 1A–D and 5B–G) and a lowdensity in the arcuate nucleus (Fig. 1C and D) and in theventromedial hypothalamic nucleus (Fig. 1C). No immunoreactivecell bodies were found in the anterior and posterior parts of theventromedial hypothalamic nucleus (Fig. 1B and D), but theimmunoreactive cell bodies observed in the other two hypotha-lamic nuclei extend from the rostral-most part to the caudal-mostpart of each nucleus. Immunoreactive fibers were found through-out the alpaca thalamus and hypothalamus (Fig. 1 and Table 1).Thus, for example, in the thalamus immunoreactive fibers werefound in nuclei belonging to the midline, medial, intralaminar,ventral and dorsal groups, whereas in the hypothalamus thesefibers were observed in nuclei located in the mamillary,periventricular, medial and lateral regions. In sum, 19 nuclei (11

Table 1Distribution of immunoreactive fibers and cell bodies containing somatostatin-28

(1–12) in the alpaca diencephalon.

SOM-28 (1–12)

Nuclei CB F

AD � +

AHy � +++

AM � ++

Arc + +++

AV � +

CL � ++

CM � +++

DA � +++

Around Fx � +++

LD � +/+++

LG � +

LH +++ +++

LHb � +++

LM � +++

LP � +

MD � +++

ME � +++

MG � +

MHb � +

MM � +

PC � ++

PF � +

PH � +++

Po � ++

PV � +++

PVH � +++

Re � +++

Rh � +++

Rt � ++

SCh � +++

SO � +++

SPF � +++

STh � ++

VA � ++

VL � ++

VM � ++

VMH + +++

VPL � ++

VPM � ++

ZI � ++

CB: cell bodies (+++: high density; +: low density). F: fibers (+++: high density; ++:

moderate density; +: low density). For nomenclature of the nuclei, see list of

abbreviations.

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–98 91

hypothalamic and 8 thalamic) of the alpaca diencephalon showeda high density of somatostatin-28 (1–12)-immunoreactive fibers. Amoderate density of such fibers was also found in 12 diencephalicnuclei, and a low density was found in 9 of them (see Table 1). Forexample, a high density of immunoreactive fibers was observed inthe anterior hypothalamic area (Figs. 1A and 4C and D), arcuatenucleus (Fig. 1C and D), dorsal hypothalamic area (Figs. 1C and Dand 3B), around the fornix (Figs. 1A–D and 3E and F), lateralhypothalamic area (Fig. 1A–E), lateral habenular nucleus (Figs. 1Eand 2C and F), mediodorsal thalamic nucleus (Fig. 1B–E),paraventricular thalamic nucleus (Figs. 1A–E and 2B), medianeminence (Fig. 1C and D), paraventricular hypothalamic nucleus(Fig. 1A and B and 3C and D), suprachiasmatic nucleus (Fig. 1A),supraoptic hypothalamic nucleus (Figs. 1A and B and 4B), and inthe ventromedial hypothalamic nucleus (Fig. 1B–D and 4E and F).Moreover, a moderate or low density of immunoreactive fibers wasfound in many alpaca diencephalic nuclei (see Table 1 and Fig. 1).

In general, in all the animals used in this study both thedistribution and density of the immunoreactive structuresobserved in the alpaca diencephalon were fairly similar. In general,the cell bodies observed were round or fusiform, small or mediumsize, showing one-to-three short/medium-length processes (longprocesses were also observed) (Fig. 5C–G), whereas the vast

majority of the immunoreactive fibers were thin, short in length,non-branched, and with varicosities (Figs. 2–4). In general, thesefibers did not show any special orientation.

4. Discussion

This is the first time that the distribution of immunoreactivestructures containing a neuropeptide has been described not onlyin the alpaca diencephalon, but also in the diencephalon ofcamelids. Immunoreactive fibers containing somatostatin-28 (1–12) were widely distributed throughout the diencephalon of thecamelid studied here, but the presence of immunoreactive cellbodies was quite restricted.

Regarding the distribution of somatostatin-28 (1–12) in the ratdiencephalon, our results are in agreement with previous reportscarried out in the rat (Benoit et al., 1982, 1985). Thus, in the rodentthose authors detected somatostatin-28 (1–12) by radioimmuno-assay in both thalamus and hypothalamus, the content of thepeptide being higher in the hypothalamus than in the thalamus; inthe hypothalamus somatostatin-28 (1–12) was highly concentrat-ed in nerve terminals, and a plexus of somatostatin-28 (1–12)-immunoreactive fibers was found in the median eminence (Benoitet al., 1982, 1985). However, in the rat periventricular nucleus,somatostatin-28 (1–12)-immunoreactive cell bodies were ob-served (Guy et al., 1985), but not in the alpaca. Moreover, it seemsthat in general the distribution of immunoreactive fibers contain-ing somatostatin-14 in the rat diencephalon (Bennet-Clarke et al.,1980; Finley et al., 1981; Johansson et al., 1984; Palkovits, 1988;Vincent et al., 1985; Willoughby et al., 1995) is quite similar to thatfound in the alpaca for somatostatin-28 (1–12) (in both speciesimmunoreactive fibers were found throughout the hypothalamusand the thalamus (midline, medial and lateral)), but the distribu-tion of cell bodies containing somatostatin-14 is more widespreadin the rat (Bennet-Clarke et al., 1980; Ceccateli et al., 1986; Finleyet al., 1981; Fitzpatrick-McElligott et al., 1988; Guy et al., 1985;Johansson et al., 1984; Palkovits, 1988; Priestley et al., 1991;Vincent et al., 1985; Willoughby et al., 1995) than the distributionof somatostatin-28 (1–12)-immunoreactive cell bodies in thealpaca diencephalon. This discrepancy is not due to the adminis-tration of colchicine, since in rats not treated with the drug (seeWilloughby et al., 1995), the distribution of somatostatin-14-immunoreactive cell bodies was more widespread that that foundin the alpaca for somatostatin-28 (1–12). Moreover, it has beenreported that the distribution of prosomatostatin mRNA-contain-ing cell bodies is coextensive with the location of neuronscontaining somatostatin-28 (Fitzpatrick-McElligott et al., 1988).In both cases, the distribution of prosomatostatin mRNA- orsomatostatin-28-containing perikarya was more widespread thatthat found for somatostatin-28 (1–12)-immunoreactive cell bodiesin the alpaca thalamus and hypothalamus.

Comparing our data with those reported on the distribution ofthe immunoreactive fibers containing somatostatin-28 (1–12) inthe cat diencephalon (de Leon et al., 1991), it seems that theirdistribution is widespread in the alpaca. In both the cat and alpacasomatostatin-28 (1–12)-immunoreactive fibers were observedthroughout the hypothalamus and in the midline thalamic nuclei.However, an important difference was observed. In the feline(treated and not treated with colchicine), the lateral and somemedial thalamic nuclei are devoid of immunoreactive structurescontaining somatostatin-28 (1–12), whereas in the camelid thisimmunoreactivity was found in those thalamic nuclei. It seemsthat these differences regarding the distribution of the immuno-reactive structures containing somatostatin-28 (1–12) in thelateral and medial thalamus of both the cat and alpaca wouldbe due to species differences, since the same method was carriedout (e.g., fixative, first antiserum, immunocytochemical technique,

Fig. 1. Distribution of somatostatin-28 (1–12)-immunoreactive structures in frontal planes of the alpaca diencephalon from rostral (A: anterior hypothalamic area) to caudal

(E: medial mammilary nucleus) levels. Cell bodies containing such neuropeptide are represented by closed circles (high density) and squares (low density), whereas

immunoreactive fibers are represented by lines with varicosities (low density), continuous lines (moderate density) and crossed lines (high density). For nomenclature of the

nuclei, see list of abbreviations.

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–9892

mapping) in both species (de Leon et al., 1991). However, regardingthe distribution of cell bodies containing somatostatin-28 (1–12)in the cat and alpaca thalamus and hypothalamus, it seems that thedistribution of such cells is more widespread in the feline (de Leonet al., 1991). However, an important difference should be noted; inthe alpaca we observed cell bodies containing somatostatin-28 (1–12) in three hypothalamic nuclei (arcuate nucleus, lateralhypothalamic area, ventromedial hypothalamic nucleus), but suchnuclei containing somatostatin-28 (1–12)-immunoreactive peri-karya have not been found in the cat (de Leon et al., 1991). All thesedata suggest that the distributions of immunoreactive cell bodiescontaining the neuropeptide in the cat and alpaca diencephalonare quite different. This may be due to technical considerations,since in the cat colchicine was administered to the animals (deLeon et al., 1991), whereas in the alpaca we used animals that were

not treated with the drug. In this sense, further research would benecessary to discover the origin of the discrepancies mentioned inthe cat and alpaca thalamus and hypothalamus regarding thedistribution of fibers and cell bodies containing somatostatin-28(1–12). In comparison with a previous study carried out on thedistribution of somatostatin-14-immunoreactive structures in thecat diencephalon (Graybiel and Elde, 1983), it seems that thedistribution of somatostatin-28 (1–12)-immunoreactive fibers inthe alpaca caudal diencephalon is more widespread than thatfound for somatostatin-14-immunoreactive fibers in the samediencephalic region of the cat. However, cell bodies containingsomatostatin-14 were located in the cat reticular thalamic nucleus(Graybiel and Elde, 1983), but in this nucleus no immunoreactivecell bodies containing somatostatin-28 (1–12) were found in thealpaca.

Fig. 2. Somatostatin-28 (1–12)-immunoreactive fibers in the alpaca thalamus. (A) Frontal section of the alpaca diencephalon. For nomenclature of the nuclei, see list of

abbreviations. The photographs shown in B–E were respectively taken from the regions delimited by the rectangles in A (indicated as B–E). (B) Immunoreactive fibers

(arrows) in the paraventricular thalamic nucleus (PV). V: ventricle. (C) Low-magnification of fibers containing somatostatin-28 (1–12) located in the lateral habenular nucleus

(LHb). s: stria medullaris. (D) Paraventricular thalamic nucleus after preabsorption of the first antibody. Note the absence of immunoreactivity. (E) Lateral habenular nucleus

after the omission of the first antibody. Note the absence of immunoreactivity. MHb: Medial habenular nucleus. (F) High-magnification of the region delimited by a rectangle

in C. Scale bar: A (1 cm); B, C, E (100 mm); D, F (50 mm).

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–98 93

Fig. 3. Somatostatin-28 (1–12)-immunoreactive fibers in the alpaca hypothalamus. (A) Frontal section of the alpaca diencephalon. For nomenclature of the nuclei, see list of

abbreviations. The photographs shown in C and E were respectively taken from the regions delimited by the rectangles in A (indicated as C and E). (B). Fibers containing the

neuropeptide (arrows) located in the dorsal hypothalamic area (DA). V: ventricle. This photograph was taken from the level shown in Fig. 1D. D: dorsal; M: medial. (C) Low-

magnification of fibers containing somatostatin-28 (1–12) located in the paraventricular hypothalamic nucleus (PVH). V: ventricle. (D) High-magnification of the region

delimited by a rectangle in C. (E) Low-magnification of the region around the fornix (Fx). (F) High-magnification of the region delimited by a rectangle in E. Scale bar: A (1 cm);

B, C, E (100 mm); D, F (50 mm).

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–9894

Fig. 4. Somatostatin-28 (1–12)-immunoreactive fibers in the alpaca hypothalamus. (A) Frontal section of the alpaca diencephalon. For nomenclature of the nuclei, see list of

abbreviations. The photograph shown in E was taken from the region delimited by the rectangle in A (indicated as E). (B) Fibers containing somatostatin-28 (1–12) (arrows)

located in the supraoptic hypothalamic nucleus (SO). ox: optic chiasm. This photograph was taken from the level shown in Fig. 1A. D: dorsal; M: medial. (C) Low-

magnification of fibers containing somatostatin-28 (1–12) located in the anterior hypothalamic area (AHy). This photograph was taken from the level shown in Fig. 1A. D:

dorsal; M: medial. (D) High-magnification of the region delimited by a rectangle in C. (E) Low-magnification of the ventromedial hypothalamic nucleus (VMH). V: ventricle.

(F) High-magnification of the region delimited by a rectangle in E. Scale bar: A (1 cm); B–F (100 mm).

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–98 95

Fig. 5. Somatostatin-28 (1–12)-immunoreactive cell bodies in the alpaca hypothalamus. (A) Frontal section of the alpaca diencephalon. For nomenclature of the nuclei, see list

of abbreviations. The photographs shown in B, E, F and G were respectively taken from the regions delimited by the rectangles in A (indicated as B, E, F and G). (B) Low-

magnification of the lateral hypothalamic area (LH). ci: capsula interna. (C) A high-magnification of the region delimited by the left rectangle in B. Arrows indicate

immunoreactive cell bodies. (D) A high-magnification of the region delimited by the right rectangle in B. Immunoreactive cell bodies are indicated by arrows. (E–G)

Immunoreactive cell bodies containing somatostatin-28 (1–12) located in the lateral hypothalamic area (LH). Scale bar: A (1 cm); B–G (100 mm).

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–9896

R. Covenas et al. / Journal of Chemical Neuroanatomy 42 (2011) 89–98 97

Our results are in general in agreement with those reported forthe regional distribution of somatostatin in the bovine brain byradioimmunoassay (Barden et al., 1981). In that work, the peptidewas observed in the thalamus and hypothalamus of both species.However, the distribution of somatostatin-28 (1–12)-immunore-active cell bodies in the sheep hypothalamus (Scanlan et al., 2003)is more widespread than that found for somatostatin-28 (1–12)-immunoreactive perikarya in the alpaca diencephalon. It should beremarked that in both species colchicine was not administered(Scanlan et al., 2003). This suggests that the differences regardingthe distribution of somatostatin-28 (1–12)-immunoreactive cellbodies in both ruminants, sheep and alpaca, would be due tospecies differences. Moreover, in the sheep hypothalamus it hasbeen reported that all neurons immunoreactive for somatostatin-14 are also immunoreactive for somatostatin-28 (1–12) (Scanlanet al., 2003). This means that in this ruminant, both somatostatinpeptides are not selectively produced by sheep hypothalamicneurons and that preprosomatostatin is not preferentially cleavedinto either somatostatin-14 or somatostatin-28 (1–12).

Moreover, it has been reported that the distribution ofsomatostatin 14-immunoreactive cell bodies is more widespreadin the sheep hypothalamus (in animals treated or not treated withcolchicine) (Papadopoulos et al., 1986; Willoughby et al., 1995)than in the alpaca hypothalamus. Thus, in the sheep immunoreac-tive cell bodies were found in the lateral hypothalamic area,arcuate nucleus, ventromedial hypothalamic nucleus, paraventri-cular hypothalamic nucleus, suprachiasmatic nucleus, supraoptichypothalamic nucleus, anterior hypothalamic area, etc. (Papado-poulos et al., 1986). In the sheep, a distinctive shell of immunore-active perikarya was observed around the ventromedialhypothalamic nucleus and it seems that this shell of somatostat-in-14 perikarya is a significant species characteristic (seeWilloughby et al., 1995). In the alpaca, we did not observed sucha structure. Somatostatin-14-immunoreactive fibers have beenobserved throughout the sheep hypothalamus (Willoughby et al.,1995), as we have found for somatostatin-28 (1–12)-immunore-active fibers in the alpaca diencephalon. Our results are inagreement with those reported by Papadopoulos et al. (1986) inthe sheep thalamus, since these authors failed to find anyimmunoreactive cell body containing somatostatin-14 in thisdiencephalic region.

Finally, by in situ hybridization a widespread distribution ofpreprosomatostatin-labeled cells has been reported in the sheephypothalamus (Bruneau and Tillet, 1998), this distribution being ingood agreement with that reported for somatostatin-14/somato-statin-28-immunoreactive cell bodies (Papadopoulos et al., 1986;Scanlan et al., 2003; Willoughby et al., 1995), and it is also knownthat the distribution pattern of somatostatin receptors in the sheepbrain is closer to the situation in human than in rodents (Fodoret al., 1997).

Regarding the distribution of somatostatin-14/somatostatin-28in the human diencephalon (Bennet-Clarke and Joseph, 1986;Bouras et al., 1987), it appears that in general the distribution of theimmunoreactive fibers containing the tetradecapeptide or so-matostatin-28 is similar to that seen in the alpaca for somatostat-in-28 (1–12), but the distribution of somatostatin-14/somatostatin-28-immunoreactive cell bodies is more widespreadin the human than in the alpaca diencephalon for perikaryacontaining somatostatin-28 (1–12). For example, immunoreactiveperikarya containing somatostatin-28 have been observed in thesuprachiasmatic nucleus, supraoptic nucleus, paraventricularhypothalamic nucleus, arcuate nucleus, lateral hypothalamic area,ventromedial hypothalamic nucleus, dorsal hypothalamic area andin the reticular thalamic nucleus. In this thalamic nucleus,perikarya containing prosomatostatin-derived peptides have beendescribed in cat, monkey and human (Bouras et al., 1987; Graybiel

and Elde, 1983), but not in the rat (Bennet-Clarke et al., 1980;Finley et al., 1981; Johansson et al., 1984; Palkovits, 1988; Vincentet al., 1985) or alpaca. Our results are in agreement with thosefound for the thalamus and hypothalamus of humans (Cooperet al., 1981). By means of radioimmunoassays, the presence ofsomatostatin has been reported in both central nervous systemregions. In the human hypothalamus, those authors found a higherconcentration of somatostatin in the hypothalamus than in thethalamus. This is also in agreement with our results, since wefound more nuclei in the alpaca hypothalamus containing a highdensity of immunoreactive fibers than in the thalamus and in thehypothalamus only one nucleus (the medial mammilary nucleus)showed a low density of immunoreactive fibers, whereas thethalamus had several nuclei with a low density of immunoreactivefibers.

The discrepancies in the location of different forms ofsomatostatin (somatostatin-14, somatostatin-28) in the samenuclei/region of the mammalian central nervous system may bedue to intraneuronal transport of the peptides, to a differentialpost-translational processing of pre-prosomatostatin to producedifferent end products, or to a different ratio of the peptidergicfragments and/or due to problems of sensitivity (see de Leon et al.,1991).

To conclude, we have reported for the first time the presence ofimmunoreactive fibers and cell bodies containing somatostatin-28(1–12) in the diencephalon of a camelid. Immunoreactivestructures containing the neuropeptide were widely distributedthroughout the diencephalon of the alpaca. This widespreaddistribution indicates that somatostatin-28 (1–12) could beinvolved in many physiological functions in the alpaca diencepha-lon. The diencephalic distribution of somatostatin-28 (1–12)-immunoreactive structures described here in the alpaca dienceph-alon suggests that the peptide would be involved in feeding, sexualactivity, vigilance and attentive and affective defence behaviours,homeostasis, thermogenesis, circadian rhymths and in neuroen-docrine, visual and stress mechanisms (Bouyer et al., 1992;Covenas et al., 1993; Fuchs et al., 1985; Pickard, 1985; Prestonet al., 1989; Sheward et al., 1984). Future studies should focus onsuch possible functions.

Acknowledgements

This work has been supported by the Ministerio de Educacion yCiencia (BFU2005-02241/BFI), Spain and the Ministerio de Cienciae Innovacion (BFU2008-03369/BFI), Spain and by the CONCYTEC:PROCYT project 2006, Peru. The authors thank N. Skinner forstylistic revision of the English text and Professor Gerard Tramu(University of Bordeaux I, France) for kindly providing thesomatostatin-28 (1–12) antiserum.

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