Brain Struct Funct DOI 10.1007/s00429-014-0928-0

ORIGINAL ARTICLE

Neurotensin: revealing a novel neuromodulator circuit in the nucleus accumbens–parabrachial nucleus projection of the domestic chick Eszter Ba´lint • Tama´s Bala´zsa • Gergely Zachar Szilvia Mezey • Andra´s Csillag

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Received: 29 July 2014 / Accepted: 18 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Lower brainstem projections from nucleus accumbens (Ac) subregions to the parabrachial complex (PB), the nucleus of the solitary tract and the vagal motor nuclei have been described previously in the domestic chick by our group. Such projections, particulary those from the core and rostral pole regions of Ac have not been found in mammals or pigeons. Here we report on the presence of neurotensin (NT) in the neurons projecting from different Ac subnuclei, and also from the bed nucleus of stria terminalis, to the PB in the domestic chicken. The study is based upon correlated retrograde tracing (using Fast Blue) and NT immunohistochemistry, supplemented with regional charting and quantitative analysis of doublelabeled neurons. The number of retrogradely labeled cells in Ac subdivisions reflects the size of FB tracer deposit, and the degree to which it extends to the medial PB. Of all Ac subregions, the core contained the largest amount of double-labeled cells. The findings demonstrate that the anatomical pathway through which the Ac can directly modulate taste-responsive neurons of the PB employs mainly neurotensin as a neuromodulator. The observed anatomical difference between mammals and birds is either a general taxonomic feature or it reflects feeding strategies specific for the domestic chick. The results are also relevant to a better understanding of the role of NT in food intake and reward-related behaviors in birds. Keywords Basal ganglia
Brainstem
Neuropeptides
Taste processing
Avian brain
Pathway tracing

E. Ba´lint (&)
T. Bala´zsa
G. Zachar
S. Mezey
A. Csillag Department of Anatomy, Histology and Embryology, SemmelweisUniversity, 58. Tuzolto utca, 1094 Budapest, Hungary e-mail: [email protected]

Introduction Like in mammals, the nucleus accumbens (Ac) of domestic chicks consists of three subdivisions: the rostral pole (AcR) is located in the rostral part of the basal telencephalon, the core (AcC), corresponding to the region previously described as the ventromedial medial striatum (MSt), and the shell (AcS), positioned ventrally and ventrolaterally to the AcC (Balint and Csillag 2007). The projections of Ac subregions in the domestic chick are largely similar to those of mammals, including projections to the ventral pallidum, lateral hypothalamus, preoptic area and dopaminergic brainstem nuclei (ventral tegmental area and substantia nigra, pars compacta) (Balint et al. 2011, Groenewegen et al. 1999; Husband and Shimizu 2011; Troiano and Siegel 1978; Zahm and Heimer 1993). However, there are some notable differences: both the AcC and AcR of the domestic chick send fibers to the parabrachial complex (PB, more to the medial parabrachial nucleus, MPB, less to the lateral parabrachial nucleus, LPB), the nucleus of the solitary tract (NTS), and the vagal motor nuclei (Balint et al. 2011). These visceral projections have not been described in mammals or pigeons (Groenewegen et al. 1999; Husband and Shimizu 2011; Usuda et al. 1998; Zahm and Heimer 1993). However, projections from the AcS to the parabrachial nuclei are present in both domestic chicks (Balint et al. 2011) and mammals (Usuda et al. 1998; Zahm et al. 1999). In mammals, theAc plays a crucial role in addiction, emotionality, goal-directed behavior and responses related to natural reinforcers (Berridge and Robinson 1998; Groenewegen and Uylings 2000; Kelley 1999, 2004). The different accumbens subregions, the AcS and AcC, are differentially involved in feeding-related processes, e.g., establishing the taste memory trace depending on the

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quality of the stimulus and association of taste with visceral consequences (Ramirez-Lugo et al. 2006, 2007). Moreover, the AcS is involved in the regulation of food intake, forming a link between cortical circuits and hypothalamic/ brainstem visceral regions. AcS directly controls feeding through its projections to the lateral hypothalamus (LH, Kelley 2004; Stratford and Kelley 1999), codes the hedonic value of food (Berridge et al. 2009; Hajnal et al. 2004), and also plays an important role in regulating behavior in response to palatable stimuli (Di Chiara 2002; Loriaux et al. 2011; Norgren et al. 2006). Thus, while the AcS is driven primarily by unconditioned visceral stimuli, similar to the extended amygdala in general, the AcC is engaged in the processing of learning-related conditioned stimuli and the execution of adaptive instrumental actions, much like the dorsal striatum (Corbit et al. 2001; Ramirez-Lugo et al. 2007). In mammals, the second central relay for the gustatory pathway, the parabrachial nucleus, receives gustatory information from the NTS, transferring it to various forebrain gustatory nuclei (bed nucleus of the stria terminalis, pars lateralis, BSTl, central amygdaloid nucleus and the LH), as well as—via the thalamic ventral posteromedial nucleus—to the gustatory cortex (Lundy and Norgren 2004a). In birds, PB also receives gustatory and visceral sensory information from the NTS (Arends et al. 1988; Berk et al. 1993; Wild et al. 1990), and projects to various forebrain regions, including the LH, BSTl, the amygdaloid arcopallium and, mainly, the core subregion of Ac (Wild et al. 1990). Even though the latter projection was not specified in the cited paper, its presence is evident from the relevant figure (Fig. 2 in Wild et al. 1990). A series of studies demonstrate that the mammalian PB is crucial for assigning hedonic value to taste stimuli, enabling these to activate the reward system (Hajnal et al. 2009; Mungarndee et al. 2008; Norgren et al. 2006). Concentration-dependent dopamine release in the Ac after sham sucrose-licking can be blunted after PB lesions (Hajnal et al. 2009). Similarly, c-Fos activation in the AcS after sucrose ingestion can be prevented by lesions of PB (Mungarndee et al. 2008). Ventral forebrain areas associated with affective responses to taste stimuli appear to be activated directly by PB gustatory neurons rather than via the thalamocortical taste system (Mungarndee et al. 2008). Taste-responsive neurons in the PB are modulated by so-called rostral gustatory nuclei: electrical stimulation of the LH, central amygdala, BSTl and the AcS modifies neuronal firing of PB-neurons in response to taste stimuli, in rats and hamsters, regardless of the type of taste stimulus (Cho et al. 2003; Li and Cho 2006; Li et al. 2002, 2005, 2012; Lundy and Norgren 2001, 2004b). This forebrain modulation of pontine taste relay is crucial in learned taste-

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guided behavior, e.g., conditioned taste aversion (Grill 1985). Widely distributed throughout the central and peripheral nervous systems, the 13-amino-acid peptide neurotensin (NT) is highly conserved among mammals and birds (Alexander et al. 1989; Atoji et al. 1996; Carraway and Bhatnagar 1980; Esposito et al. 1997; Goedert et al. 1985; Reinecke 1985). In both taxa, NT-containing cells are present in large number in the Ac, the neighboring dorsal striatum and BSTl (Atoji et al. 1995b, 1996; Jennes et al. 1982; Zahm 1987). These cells are projection neurons sending their fibers to the globus pallidus, ventral tegmental area, substantia nigra and retrorubral field (Sugimoto and Mizuno 1987). NT contains a 6-amino-acid active sequence at the C-terminal (identical in mammals and birds), responsible for its biological role (Vincent et al. 1999). This sequence is quite similar across all peptides (NT, neuromedin-N and LANT6) derived from the same 170-amino-acid precursor protein (Carraway et al. 1993a, b; Dobner et al. 1987). However, in monkeys and birds, LANT6 is only present in a subpopulation of striatal interneurons that are larger than typical medium spiny neurons (Reiner and Anderson 1993). Neurotensin fibers densely innervate feeding-related brainstem regions including the PB (Atoji et al. 1996; Milner and Pickel 1986),with proven synaptic interactions with dendrites and axon terminals in the neuropil of PB (Milner and Pickel 1986). This, likely, forms the anatomical substrate for NT modulation of synaptic transmission, e.g., enhancing evoked postsynaptic currents, as described in rat pontine slices (Saleh et al. 1997). The aim of the present study was to reveal the presence of NT in the neurons projecting from different Ac subnuclei and the BSTl to the PB in the domestic chicken. The findings are relevant to the better understanding of the role of NT in food intake and reward-related behaviors in birds.

Materials and methods Animals Six 10-day-old Hunnia broiler hybrid domestic chickens were used for the retrograde pathway-tracing experiments. Food and water were available ad libitum. The experiments were conducted in conformity with the laws and regulations controlling experiments and procedures in live animals, as described in the Principles of laboratory animal care (NIH Publication No. 85-23, revised 1985) and the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (protocols ETS No. 170ETS No.123).

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Retrograde tracer injection The animals were deeply anesthetized with a mixture containing 34 mg/kg body weight (b.wt.) ketamine and 7 mg/kg b.wt. xylazine (i.m.). Then, 0.2 ll of the retrograde tracer Fast Blue (FB, Polysciences, Warrington, PA; 5 %, dissolved in distilled water) was injected stereotaxically into the parabrachial region (ML: -0.26 cm, AP: -0.26 cm, DV: -0.75 cm; beak bar: -0.5 cm, Kuenzel and Masson 1988), using a 1.0 ll Hamilton syringe mounted on a Kopf microinjector unit. The tracer was deposited by slow pressure injection lasting 10–15 min. The needle was only retracted after a 15-min resting interval, to avoid leakage of FB along the injection track. Perfusion and sectioning After 5 days of survival the chicks were deeply anesthetized (as above) and transcardially perfused first with 100 ml saline followed by 500 ml 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed from the skull and postfixed at 4 °C in 4 % paraformaldehyde in 0.1 M phosphate buffer overnight, then transferred to 30 % sucrose in phosphate-buffered saline (PBS; 4 °C, 2 days). Brains were sectioned at 60 lm on a Leica freezing microtome in the coronal plane. Sections were stored at 4 °C in 0.02 % sodium azide in PBS until further processing for histochemistry. Immunohistochemistry Of the six experimental birds, five animals were further processed for immunohistochemistry. Tissue sections were rinsed in phosphate-buffered saline (PBS) containing 0.05 % Tween-20 (20 min, 49). To avoid non-specific labeling, sections were rinsed in blocking serum (5 % normal donkey serum in PBS) for 60 min at room temperature. Specimens were incubated for 48 h at 4 °C in PBS containing the anti-neurotensin primary antibody (1:1,000, made in rabbit, Peninsula Laboratories). Sections were washed in PBS (49), incubated for 3 h in PBS containing Alexa 488-conjugated anti-rabbit IgG (1:250 in PBS), rinsed in PBS (49), and mounted in glycerol–PBS (mixture of 1:1) mounting medium. Neurotensin antibody The rabbit polyclonal anti-neurotensin primary antibody (Peninsula Laboratories, cat. no. T-4313) was raised against bovine neurotensin 1–13 residues and characterized and specified by the manufacturer. The specificity of the antibody has been studied using ELISA in earlier studies

(Atoji et al. 1994, 1995a). The extent of the cross-reactivity between neurotensin and LANT-6 was 1.6 % in those experiments. The pattern of NT staining observed in the present study was consistent with that of previous studies, which had examined the NT distribution in the brain of domestic chick, pigeon (Atoji et al. 1996), and rat (Zahm 1987). Given the expected pattern and a sufficient degree of perikaryal labeling, colchicine pretreatment was not considered to be necessary, even if the amount of NT neurons colocalizing with FB might be slightly underestimated. Immunocontrols In control sections, the primary antibodies were replaced by pre-immune normal serum. No visible signal was detected when these sections were processed simultaneously with experimental sections through identical incubation steps (see also the statement on negative control under Western blotting). Western blotting Striatal samples from 2-week-old domestic chicks were homogenized in ice-cold lysis buffer (150 mM NaCl; 1 % Nonidet P-40; 0.5 % sodium deoxycholate; 0.1 % sodium dodecyl sulfate, SDS; 50 mM Tris, pH 8.0). Insoluble material was removed by centrifugation (15,000g, 10 min, 4 °C). Protein concentrations in the brain homogenates were determined using the BCA method (Olson and Markwell 2007). Samples were dissolved in Laemmli buffer (Sigma-Aldrich) and boiled for 5 min. Samples containing 5 lg of total protein were loaded onto 12 % acrylamide gels and processed with BioRad Mini-Protean III vertical electrophoresis and blotting systems. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and electrophoretically transferred to nitrocellulose membranes. Then, the membranes were immediately incubated with 1 % skimmed milk (Cell Signaling) in TBS–T buffer (0.05 M Tris-buffered saline pH 7.4 and 0.1 % Triton X) for 1 h at room temperature, followed by an incubation with rabbit polyclonal anti-neurotensin (Peninsula Laboratories, cat. no. T-4313), at a dilution of 1:4,000 and 1:8,000 in TBS–T buffer, containing 1 % skimmed milk (Cell Signaling), at 4 °C overnight. For detection, the membranes were incubated with HRP-conjugated anti-rabbit polyclonal secondary antibody (1:20,000, DAKO) and the blots were visualized with an enhanced chemiluminescence detection system (Immobilon Western Chemiluminescent HRP Substrate; Millipore). Since the molecular weight of NT itself falls below the detectability level of Western blotting, we could only

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Statistical analysis Injections were ranked on an ordinal scale from 1 to 5 according to their relative size. Cell counts were correlated to injection size ranks using Spearman’s nonparametric correlation. The cell counts were compared among the five observed brain regions using Friedman test. Post hoc pairwise comparisons were carried out using Wilcoxon signed-rank test.

Fig. 1 Western blots for neurotensin antibody. Five micrograms of total protein from four 2-week-old domestic chicks were separated on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes by Western blotting, and exposed to anti-neurotensin at a dilution of 1:4,000 (lanes 1 and 2) and 1:8,000 (lanes 3 and 4)

detect the known precursor molecule of the neurotensin/ LANT6 (Carraway et al. 1993a). The presence of the bands at 17 kDa molecular weight (Fig. 1), which corresponds to the known neurotensin precursor molecule supports the specificity of the antibody employed in our study. When the primary antibody was omitted, the respective band of the gel came out blank (not shown). Microscopy and quantitative analysis Selected sets of microscopic specimens from one extra bird (not used for confocal laser microscopy) were set aside for fluorescent microphotography. Distribution of retrogradely labeled neurons was examined using an Olympus BX-51 fluorescent microscope equipped with a digital camera, using the image capturing programs Viewfinder Lite and Studio Lite. Photomicrographs were compared with adequate brain atlas charts (Kuenzel and Masson 1988; Puelles et al. 2007) to define the position of labeled cells. For quantification, retrogradely labeled, Fast blue-containing (FB?) and neurotensin-immunopositive (NT?) cell bodies were visualized with a laser-scanning confocal microscope (Nikon-BioRad Eclipse E800). To quantify the percentage of NT? neurons among the cells projecting to the parabrachial nuclei (FB?), we counted the NT? and FB? neurons in the entire volume of all accumbens subregions and neighbouring BSTl. Double labeling was examined using confocal Z-stack images spanning the entire thickness of one 60 lm section. All retrogradely labeled (FB?) neurons and all FB-NT double-labeled cells were counted across the whole depth of the section using the Adobe Photoshop CS5 software. For schematic diagrams, we used the Adobe Illustrator CS5 vector-based illustration software.

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Results Injection sites Injections of FB resulted in deposit sites of variable size in the PB (Fig. 2). In case 1, the injection hit the rostral PB, the ventral part of MPB and LPB (Fig. 2a–c). The tracer spread between IA-2.24 and IA-2.96 mm, almost the whole rostrocaudal extent of PB. In case 2, the injection affected the dorsal PB (DPB) and the rostral part of MPB (Fig. 2d). One injection (case 3) was placed in the dorsal part of MPB, but affected the DPB, the caudal, ventral MPB and the LPB (Fig. 2e–f). The tracer spread between IA-2.48 and IA-2.96 mm. In case 4, the tracer affected only a very small region in the caudal part of the ventral LPB and lateral MPB (Fig. 2g). A relatively larger injection, case 5 affected the central and caudal part of MPB with the tracer spreading into the LPB (Fig. 2h). In case 1 and 3, a small amount of tracer also spread into area 7. Distribution of retrogradely labeled neurons Retrogradely labeled cells were always observed in the ipsilateral hemisphere only. FB? neurons were differentially distributed in the ventrobasal forebrain depending on the injection site. The density of labeled cells tended to diminish in a medial to lateral direction (Fig. 3). The number of retrogradely labeled (FB?, regardless of NTcontent) and double-labeled cells, as well as the ratio of double-labeled cells to all FB? neurons in the AcC, AcS and BSTl correlated with the size of the injection as revealed by Spearman’s rank correlation test (Table 1; Fig. 4). In the case of AcR, the number of FB? cells did not correlate with the injection size, however, there was a strong correlation between the number or the ratio of double-labeled cells and the injection size. Friedman test revealed differences among the four brain regions in the number of FB ? cells (v2 = 16.0, df = 4, p = 0.003), double-labeled cells (v2 = 14.7, df = 4, p = 0.005) and the ratio of double-labeled cells to all FB? neurons (v2 = 11.0, df = 4, p = 0.027). Generally, the highest number of FB ? neurons was found in the BSTl

Brain Struct Funct Fig. 2 Schematic illustration of individual BDA injections deposited in the parabrachial nuclei and surrounding regions. a–c Case 1; d case 2; e–f case 3; g case 4; h case 5. A7D dorsal A7 noradrenaline cells, DPB dorsal parabrachial nucleus, IA interaural, LPB lateral parabrachial nucleus, mlf medial longitudinal fasciculus, MPB medial parabrachial nucleus, Pr5R principal sensory trigeminal nucleus, rostral part, scp superior cerebellar peduncle, unc uncinate fasciculus

(Table 1; Figs. 3, 5a). The number of FB? cells in the BSTl was significantly greater than the number of FB? neurons in the AcR, AcS but not AcC (Fig. 5a, Wilcoxon signed-rank test, p = 0.043). In the Ac, retrogradely labeled cells were present in moderate to high number in the AcC (Table 1; Figs. 3, 4a). The AcC contained a significantly larger number of FB? cells than AcS and AcR (Fig. 5a, Wilcoxon signed-rank test, p = 0.043). However,

in case 5, where the injection affected the caudal LPB and a part of the ventral, caudal MPB, retrogradely labeled cells showed a more uniform distribution among accumbal subregions (Table 1; Fig. 4). In case 4, where the tracer was injected into the caudal part of LPB, no FB? neurons were found in the AcS (Table 1; Fig. 3), moreover, the AcC and AcR contained almost the same number of retrogradely labeled cells.

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Brain Struct Funct Fig. 3 Representative photomicrographs depicting the distribution of FB? retrogradely labeled neurons after FB injection into the parabrachial region. In the rostral plane (a), medial part of the telencephalon contains many retrogradely labeled neurons (arrowheads) distributed throughout the AcR and BSTl. Many neurons are visible in the medial part of dorsal MSt. More caudally (b), retrogradely labeled cells are widely distributed in the AcC and BSTl. FB? neurons are more scarce in the AcS. Inset injection site in the midbrain. AcC nucleus accumbens, core, AcR nucleus accumbens, rostral pole, AcS nucleus accumbens, shell, BSTl bed nucleus of the stria terminalis, pars lateralis, DPB dorsal parabrachial nucleus, LPS pallio-subpallial lamina, MPB medial parabrachial nucleus, MSt medial striatum, VL lateral ventricle. Scale bar 500 lm

Table 1 Number of retrogradely labeled and double-labeled neurons after injections of FB into the PB Rank of injection size

AcR NT ? FB

AcC FB

Ratio

NT ? FB

AcS

BSTl

FB

Ratio

NT ? FB

FB

Ratio

NT ? FB

FB

Ratio

c1

5

142

165

0.861

1,461

1,854

0.788

227

283

0.8021

4,353

5,142

0.847

c2 c3

3 4

37 172

50 245

0.74 0.702

520 577

705 955

0.738 0.604

43 62

68 98

0.6324 0.6327

436 1,097

614 1.577

0.71 0.696

c4

1

51

86

0.593

50

101

0.495

0

0

n.d.

135

196

0,689

c5

2

82

129

0.636

92

160

0.575

29

78

0.3718

531

953

0,557

0.9

1

1

0.9

1

0.9

1

0.9

0.9

0.037

B0.01

B0.01

0.037

B0.01

0.037

B0.01

0.037

0.037

Spearman’ rho p

n.s.

n.s

n.s

c1-5 case 1–5

Distribution of neurotensinergic and double-labeled neurons The Ac subregions and BSTl were densely populated by neurotensinergic cells. Some of these NT? cells were also FB?, being retrogradely labeled from the PB (Figs. 6, 7). Similar to the FB? neurons, the number of double-labeled cells in the BSTl was significantly higher than in the AcR and AcS (Fig. 5b, Wilcoxon signed-rank test, p = 0.043).

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Among all Ac subregions, the AcC contained the largest amount of double-labeled cells (Table 1; Figs. 5b, 7b, c). This difference is significant in case of AcS, but not AcR (Fig. 5b, Wilcoxon signed-rank test, p = 0.043). The ratio of double-labeled cells to all retrogradely labeled neurons correlated with the injection size in case of AcR, AcC and AcS, but not the BSTl. There were some subregional differences in this ratio, but this difference was found to be significant only between AcC and AcR: the

Brain Struct Funct

ratio of double-labeled neurons to all FB? neurons being significantly greater in the AcR (Wilcoxon signed-rank test, p = 0.043).

Discussion

Fig. 4 Schematic diagram showing the number of FB? cells in the Ac according to different tracer injections and the distribution of retrogradely labeled neurons among the different accumbens subregions. The individual cases were lined up according to increasing injection size on the x-axis

Fig. 5 Schematic diagram showing the subregional distribution of FB? (a) and FB-NT double-labeled (b) neurons. The columns labeled with identical letters of the alphabet are not significantly different from each other

As expected, larger injection sites in the PB resulted in more retrogradely labeled cells in the Ac. However, this is only partially due to a larger amount of tracer injected and taken up by axon terminals in the PB. Larger injection sites also occupied a greater volume of the PB, revealing a distinct topographical projection pattern. In the case of the smallest injection (case 4, Fig. 2g), the injection site was restricted to a small area in the caudal, ventral part of LPB and only a small amount of tracer spread to the caudal MPB. Conversely, with the largest injection (case 1, Fig. 2a–c), the tracer spread throughout the whole rostrocaudal extent of the MPB. In other words, the more the tracer spread into the MPB (case 1, case 3) the more FB? neurons were detected in the Ac, especially in the AcC. This result is in line with our previous observation on anterograde projections arising from the Ac subregions (Balint et al. 2011). The difference in the accumbal innervation of distinct PB subregions is in agreement with the known parcellation of avian PB (Arends et al. 1988; Wild et al. 1990; Wild and Arends 1987). For example, the rostral and ventral parts of PB are known to participate in vocal and respiratory control in the pigeon, based on their afferents from the pulmonary recipient subnucleus of NTS and efferents to the hypoglossal nucleus (Wild and Arends 1987). This rostral PB region, however, comparable to the Ko¨lliker-Fuse nucleus of mammals, was not included in our pathway-tracing experiments. On the other hand, the dorsal and medial parts of avian parabrachial nucleus receive projections from the gustatory subnucleus of the NTS (mVal, Arends et al. 1988) suggesting the role of MPB in gustatory functions. The subregional distribution of retrogradely labeled cells in the Ac reported here is also in agreement with our previous experiments (Balint et al. 2011): the AcC contains significantly more neurons projecting to the PB than do the AcS or AcR. Moreover, there was a large number of FB? perikarya in the BSTl, a region sending massive projection to all subregions of the PB (Balint et al. 2011). According to the present results, the ventral forebrain of the domestic chick contains a significant number of cells which project to the PB and are also immunoreactive to neurotensin. The distribution of NT-FB double-labeled neurons (Fig. 7) follows essentially the same pattern as the distribution of all retrogradely labeled cells. However, the difference in the number of NT-FB? somata is only

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Brain Struct Funct Fig. 6 Representative confocal microscopic images showing retrogradely labeled (FB?, arrows) and double-labeled neurons (arrowheads) in the ventral forebrain. a Rostral level of BSTl and the AcR. b Caudal level of the BSTl and AcC. c Caudal level of the AcS. Scale bar 100 lm. d–f Higher magnification images of two double-labeled neurons in AcC. Scale bar 25 lm

significant between AcC and AcS (but not between AcC and AcR, although a similar trend is also evident in the latter case). Concerning the percentage of NT-FB double-labeled neurons compared to all FB neurons, the only significant difference between Ac subregions was found between the AcR and AcC: the ratio was significantly greater in the AcR than in AcC. The avian PB—like its mammalian equivalent— receives projections from various forebrain regions: the BSTl, ventral pallidum (VP), arcopallium, the medial portion of MSt, and Ac subnuclei (present results, Balint et al. 2011; Wild et al. 1990). The neurotransmitter profile of these projections, however, has not yet been reported. Based on the aforementioned, the potential role of NT could be inferred as a reasonable assumption. In mammals, the PB is densely innervated by neurotensin-containing fibers (Milner and Pickel 1986). Although some NT-containing perikarya are known to be present in the PB itself, the source of these fibers is mainly extrinsic to the nucleus. One known source of NT-containing afferents is the NTS, both in mammals and in birds (Berk et al. 1993; Milner and Pickel 1986). In addition, a massive peptidergic (e.g., neurotensinergic) innervation arises from the extended amygdala, i.e., BSTl and central amygdaloid nucleus, as well as the LH, as reported in mammals (Milner and Pickel 1986; Moga et al. 1990). These forebrain connections modulate taste-evoked

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responses in the PB (Li and Cho 2006; Li et al. 2005; Lundy and Norgren 2001, 2004b). In mammals, the PB also receives projection from the AcS but the neurochemical characteristics of this projection are unknown (Usuda et al. 1998; Zahm et al. 1999). Even though the forebrain modulation of PB is mainly inhibitory, GAD-67 was not found to be present in forebrain neurons innervating the PB (Saggu and Lundy 2008). The mammalian PB is a key region in the acquisition of conditioned taste aversion (CTA), in which the animal learns to associate a certain taste with malaise caused by a toxic substance (Garcia et al. 1955). Neurons responding to innate or conditioned aversive tastes are distributed in the central medial and external lateral subnuclei of the PB (Yamamoto et al. 2009). Albeit most birds locate food based on visual cues rather than taste, malaise is more likely to be associated with taste. Therefore, CTA can be elicited also in birds, e.g., domestic chickens (Franchina et al. 1997; Gillette et al. 1980; Hayne et al. 1996). Moreover in birds, taste cues can enhance aversion to visual cues (Clarke et al. 1979; Franchina 1997; Franchina et al. 1997; Gillette et al. 1980; Hayne et al. 1996). Considering the role of Ac in the taste aversion process, aversive conditioning in the rat induces a significant increase of c-Fos immunoreactivity in the target sites of PB projections, including Ac subregions. The Ac receives massive afferentation from the BLA (Groenewegen et al. 1999; Hanics et al. 2012) and, in turn, it sends projections

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Fig. 7 Distribution of double-labeled neurons in the ventral forebrain after FB injection into the parabrachial region (case 3). AcC nucleus accumbens, core, AcR nucleus accumbens, rostral pole, AcS nucleus accumbens, shell, BSTl bed nucleus of the stria terminalis, pars lateralis, GP globus pallidus, LPS pallio-subpallial lamina, MSt medial striatum, tsm tractus cortico-septo mesencephalicus, VL lateral ventricle, VP ventral pallidum

to the VP, LH and retrorubral field, i.e., regions that have a crucial role in oromotor regulation (Groenewegen and Russchen 1984; Usuda et al. 1998; Zahm and Heimer 1993). Since innate or conditioned aversive tastes increase the activity of Ac-neurons (Carlezon and Thomas 2009; McCutcheon et al. 2012; Roitman et al. 2010), the Ac can directly suppress feeding-related motor activity in response to aversive tastes.

In the domestic chick, the Ac (formerly described as the medial part of the lobus parolfactorius) is also known to play a crucial role in a form of aversive learning, passive avoidance (Csillag 1999; Rose 2000; Stewart and Rusakov 1995). The region including the Ac has been implicated in long-term memory storage following aversive conditioning (Patterson and Rose 1992; Rose 2000), and it is also innervated by excitatory afferents from the amygdaloid arcopallium (Hanics et al. 2012). Efferents from the Ac, suggested to suppress brainstem centers involved in the pecking response (Csillag 1999), have been proved to invade lower brainstem centers, e.g., PB and reticular formation, thus revealing a potential anatomical basis for Ac to inhibit pecking (Balint et al. 2011). Our present results, together with previous observations by Balint et al. (2011), demonstrate that the anatomical pathway through which the Ac can directly modulate tasteresponsive neurons of the PB employs neurotensin as a neuromodulator. As for the synaptic mechanisms involved, synaptic transmission in the PB is mainly glutamatergic (Saleh and Cechetto 1994). It has also been described that, at least in the case of visceral information, NT can enhance the flow of action potentials resulting from glutamatergic transmission of vagal input through the PB to the thalamus (Saleh and Cechetto 1993, 1995). This is achieved by NT acting at presynaptic neurotensin receptors located on glutamatergic terminals (Saleh and Cechetto 1995; Saleh et al. 1997) and thus enhancing the excitatory postsynaptic currents evoked by these terminals. Provided that such a mechanism could also be feasible in taste information processing, a direct modulation (enhancement) by Ac of innate or conditioned aversive taste responses in the PB is likely to occur. According to a previous study (Shimura et al. 1997), aversive conditioning enhances responses to relevant taste stimuli in the PB. The underlying mechanism of this response in the rat and, presumably, also in the domestic chick, is likely to be associated with the ventral forebrain instrumental in neurotensinergic modulation of the tasteresponsive neurons of PB. Unlike the situation observed in mammals, where only the AcS projects into the PB (Usuda et al. 1998; Zahm et al. 1999), in the domestic chick, the nucleus accumbens core is predominant among the source regions of the ventral forebrain—PB pathway, as evidenced by the present report. Further investigation including several avian species would be required to establish whether this marked anatomical difference between mammals and birds is a general taxonomic feature or it merely reflects feeding strategies specific for the domestic chick. Acknowledgments The authors wish to thank Dr. Ja´nos Hanics for valuable technical suggestions. The study was supported by a Hungarian research Grant OTKA K-109077.

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Brain Struct Funct Conflict of interest

The authors declare no conflict of interest.

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