Galectin-3 and CD44v6 isoforms in the preoperative evaluation of thyroid nodules.

PURPOSE: Thyroid cancer is the most frequently occurring endocrine malignancy; however, preoperative diagnosis of some lesions, in particular those with follicular histology, is difficult, and a consistent number of not otherwise-specified "follicular nodules" are surgically resected more for diagnosis than therapeutic purposes. In this study, we investigated whether the lectin-related molecules CD44v6 and galectin-3, the expression of which is altered during deregulated cell growth and malignant transformation, could be potential markers for improving the diagnostic accuracy of conventional cytology. MATERIALS AND METHODS: A comparative immuno-chemical and molecular analysis was performed on 157 thyroid specimens representative of normal, benign, and malignant tissues, and on 36 cytologic samples obtained preoperatively by fine-needle aspiration biopsy from nonselected patients with palpable thyroid nodules. RESULTS: Normal thyrocytes did not express galectin-3 nor CD44v6. Although the expression of CD44v6 isnegligible in thyroiditis, these molecules are variably detected in benign and malignant proliferative lesions. Interestingly, galectin-3 is never expressed in benign lesions, but it is invariably detected in cancers. A comparative evaluation of CD44v6 and galectin-3 expression in thyroid malignancies demonstrated that these molecules are coexpressed at the messenger RNA and protein level in almost all lesions. CONCLUSION: Our findings suggest that CD44v6 and galectin-3 could be potential markers to preoperatively identify malignant transformed thyrocytes. Immunodetection of these molecules on cytologic specimens obtained by fine-needle aspiration biopsy is an accurate and improved method for selecting, on a molecular basis, those nodular lesions of the thyroid gland that need to be surgically resected.

High versus low fat/sugar food affects the behavioral, but not the cortisol response of marmoset monkeys in a conditioned-place-preference task.

The effect of a high (chocolate) versus low fat/sugar (chow) food on a conditioned-place-preference (CPP) task was evaluated in marmoset monkeys. Anxiety-related behaviors and cortisol levels before and after the CPP task were also measured. Subjects were habituated to a two-compartment CPP box and then, on alternate days, had access to only one compartment during daily 15-min conditionings, for a total of 14 trials. Marmosets were provisioned with chocolate chips in the CC-paired compartment on odd-numbered trials and standard chow in the CW-paired compartment on even-numbered trials. They were then tested for preferring the CC-paired context after a 24-h interval. During the conditioning, a significantly greater amount (in kcal/trial) of chocolate was consumed than chow, yet the foraging pattern of both food types was similar. On the test trial, the time spent in the CC-paired context increased significantly compared to pre-CPP levels, yet this response was not readily predicted by baseline behavioral or cortisol levels. Also, the chocolate CPP response was positively correlated with foraging time, rather than the amount of calories consumed. The sudden absence of the food increased exploration, while the chocolate CPP effect was associated with vigilance - both anxiety-related behaviors in marmosets. This behavioral profile occurred regardless of any concomitant change or correlation with cortisol. Therefore, the high fat/sugar food was more prone to be overly consumed by the marmosets, to induce a CPP response and to lead to anxiety-related behavior in its absence.

Comparative effects of lesions to the ponto-cerebellar and olivo-cerebellar pathways on motor and spatial learning in the rat.

Emerging evidence supports the role of the cerebellum in motor learning and previous studies have also shown that olivary projections to the cerebellum are involved in motor learning. Since the pontine nuclei make up the other main relay centre in the cerebro-cerebellar pathway, the purpose of the present study was to verify the involvement of the ponto-cerebellar pathway in motor and spatial learning, by comparing these functions in intact animals and in rats with selective injury of the olivary or pontine neurons. Two groups of rats were used: the first was treated with 3-acetylpyridine to destroy the inferior olivary complex, the second received electrolytic lesions of the middle cerebellar peduncle to interrupt the ponto-cerebellar pathway. Control and lesioned rats were then submitted to three tasks: unrotated rod, rota-rod at 20 r.p.m., and Morris water maze. In the first task both 3-acetylpyridine-treated rats and rats with lesions of the middle cerebellar peduncle showed static equilibrium deficiencies. Through training, however, they reached the maximal score attained by the controls. The rats submitted to the rota-rod at 20 r.p.m. obtained scores significantly inferior to the controls. The Morris water maze results indicated that the lesion of inferior olivary complex and middle cerebellar peduncle both alter learning of the spatial task. These findings show that both the ponto- and olivo-cerebellar pathways are involved in learning complex motor sequences and spatial tasks. Since both projections converge onto Purkinje cells, our results suggest an integration of these two pathways in the cerebellar control of learning mechanism.

Spatial memory impairment induced by lesion of the mesohippocampal dopaminergic system in the rat.

The hippocampal formation has long been thought to play a role in learning and memory. Previous studies from our laboratory examined the organization of mesencephalic projections to the hippocampal formation in the rat. In order to evaluate the effects on learning and memory of retrograde selective lesions of mesencephalic dopaminergic neurons, following bilateral injection of 6-hydroxydopamine in the dorsal and ventral subiculum and adjacent CA1 field of the hippocampal formation, young adult Sprague-Dawley rats were trained in classical inhibitory avoidance, inhibitory avoidance using a multiple trial (training to criterion) and the standard Morris water maze task (cued and spatial versions). With regard to inhibitory avoidance, retention was examined one, three and 10 days after training. Concerning the Morris water maze task, 6-hydroxydopamine-lesioned and sham-operated rats received four training trials on each of four days. After training sessions, the rats were tested during a 60-s probe trial (free-swim trial) in which the platform was removed from the maze. The loss of mesencephalic dopaminergic neurons in the 6-hydroxydopamine-lesioned rats, compared to sham-operated rats, was verified by tyrosine hydroxylase immunohistochemistry. Although the 6-hydroxydopamine-lesioned rats were indistinguishable from sham-operated rats in performing the inhibitory avoidance and the cued version of the Morris water maze task, in the spatial version of the Morris water maze, lesioned rats, compared to controls, exhibited significant differences in the latency (P < 0.05), quadrant time (P < 0.01) and number of platform crossings (P < 0.05). These results suggest that the rat's ability to acquire spatial learning and memory for place navigation in the Morris water maze is likely to be dependent also on the integrity of mesohippocampal dopaminergic connections.

Serotonergic input to cholinergic neurons in the substantia innominata and nucleus basalis magnocellularis in the rat.

The aim of the present study was to determine, at the light microscopic level, whether the serotonergic fibers originating from the dorsal raphe nucleus (B7), median raphe nucleus (B8) and ventral tegmentum (B9) make putative synaptic contacts with cholinergic neurons of the nucleus basalis magnocellularis and substantia innominata. For this purpose, we utilized: (i) the anterograde transport of Phaseolus vulgaris leucoagglutinin combined with choline acetyltransferase immunohistochemistry; (ii) choline acetyltransferase/tryptophan hydroxylase double immunohistochemistry; and (iii) the FluoroGold retrograde tracer technique combined with tryptophan hydroxylase immunohistochemistry. Following iontophoretic injections of Phaseolus vulgaris leucoagglutinin in the dorsal raphe nucleus, labeling was observed primarily in the ventral aspects of the nucleus basalis magnocellularis and in the intermediate region of the substantia innominata. When Phaseolus vulgaris leucoagglutinin was combined with choline acetyltransferase immunohistochemistry, a close association between the Phaseolus vulgaris leucoagglutinin-positive fibers and cholinergic neurons was observed, even though the majority of the Phaseolus vulgaris leucoagglutinin-immunoreactive terminals seemed to establish contact with non-cholinergic elements. Following Phaseolus vulgaris leucoagglutinin injection in the median raphe nucleus, very few labeled fibers with no evident close contact with nucleus basalis magnocellularis and substantia innominata cholinergic neurons were observed. After tryptophan hydroxylase/choline acetyltransferase double immunohistochemistry, a plexus of serotonergic (tryptophan hydroxylase-positive) fibers in the vicinity of choline acetyltransferase-immunoreactive neurons of the substantia innominata and nucleus basalis magnocellularis was observed, and some serotonergic terminals have been shown to come into very close contact with the cholinergic cells. Most of the tryptophan hydroxylase-immunoreactive terminals seem to establish contacts with non-cholinergic cells. Following FluoroGold injection in the nucleus basalis magnocellularis and substantia innominata, the majority of retrogradely labeled neurons was observed mainly in the ventromedial cell group of the dorsal raphe nucleus. In this area, a minority of the FluoroGold-positive neurons was tryptophan hydroxylase immunoreactive. These findings show that serotonergic terminals, identified in very close association with the cholinergic neurons in the substantia innominata and nucleus basalis magnocellularis, derive primarily from the B7 serotonergic cell group of the dorsal raphe nucleus, and provide the neuroanatomical evidence for a direct functional interaction between these two neurotransmitter systems in the basal forebrain.

Strain-dependent effects of D2 dopaminergic and muscarinic-cholinergic agonists and antagonists on memory consolidation processes in mice.

The interaction between muscarinic-cholinergic and dopaminergic systems in the modulation of memory storage of Y-maze discrimination (YMD) task was examined in C57BL/6 and DBA/2 strains of mice. In C57BL/6 mice, post-training systemic (i.p.) administration of the D2-agonist quinpirole facilitated retention and the D2-antagonist (-)-sulpiride impaired retention. Opposite effects were observed in DBA/2 strain. The facilitating or impairing effects of quinpirole and (-)-sulpiride were blocked by simultaneous post-training administration of muscarinic-cholinergic agonists and antagonists. The memory enhancing effects of the cholinergic agonist oxotremorine were not blocked by simultaneous administration of sulpiride in C57BL/6 mice or quinpirole in DBA/2 mice. Furthermore, the memory impairing effects of the cholinergic antagonist atropine were not blocked by simultaneous administration of quinpirole in C57BL/6 mice or sulpiride in DBA/2 mice. These findings indicate that the effects of D2-receptor agonists and antagonists on retention of YMD task are strain-dependent and mediated through muscarinic-cholinergic mechanisms.

Strain-dependent involvement of D1 and D2 dopamine receptors in muscarinic cholinergic influences on memory storage.

These experiments examined the interaction of muscarinic and dopaminergic systems in influencing memory for one-trial inhibitory avoidance training in mice of the C57BL/6 and DBA/2 strains. In both strains, immediate post-training systemic administration of the muscarinic cholinergic agonist oxotremorine enhanced retention and the cholinergic antagonist atropine impaired retention. No effects were seen with injections 2 h post-training. Furthermore, the drugs did not affect retention performance of animals that received no footshock on the training trial. These results confirm previous findings indicating that muscarinic cholinergic drugs affect memory by influencing memory consolidation. In C57 mice, pretreatment with selective D1 or D2 dopamine (DA) receptor agonists (SKF 38393 or LY 171555, respectively) in otherwise non-effective doses (5 and 0.25 mg/kg, respectively) potentiated the effects of oxotremorine (0.04 mg/kg). Furthermore, in C57 mice pretreatment with selective D1 or D2 receptor antagonists (SCH 23390 or (-)-sulpiride) in otherwise non-effective doses (0.025 and 6 mg/kg, respectively) blocked the memory enhancing effects of oxotremorine. The memory impairing effects of atropine (3 mg/kg) were blocked by the D1 and D2 selective agonists and potentiated by the selective D1 or D2 antagonists. In contrast, in DBA mice, the D1 and D2 selective agonists antagonised the memory enhancing effects of oxotremorine (0.02 mg/kg) and potentiated the effects of atropine (2 mg/kg). Furthermore, the D1 and D2 antagonists potentiated the effects of oxotremorine and antagonised those of atropine. These findings indicate that although muscarinic cholinergic influences on memory storage are comparable in mice of these two strains, the cholinergic-dopaminergic interactions are opposite in the two strains. These results have implications for hypotheses of cholinergic and dopaminergic regulation of memory storage.

Mesolimbic dopaminergic neurons innervating the hippocampal formation in the rat: a combined retrograde tracing and immunohistochemical study.

A major mesolimbic projection towards the hippocampal formation (HF) has been extensively described, but no clear evidence of its dopaminergic content has been demonstrated. In order to evaluate the percentage of dopaminergic (DA) cells of ventral tegmental area (VTA-A10) and adjacent substantia nigra (SN-A9) projecting to the HF, the retrograde neuronal tracer technique was combined with the tyrosine hydroxylase (TH) immunocytochemistry. Fluoro-gold (FG) was injected in several areas (subiculum, CA1, CA3, dentate gyrus) of either septal and temporal HF. Sections containing retrogradely FG labeled neurons were either mounted directly as controls or incubated with TH antiserum and revealed with rhodamine. The quantitative evaluation of retrogradely labeled and TH-IR stained cells showed that both VTA and SN projections towards the HF are partially (15-18%) dopaminergic. Ten percent of the DA neurons of the VTA projected to contralateral HF, whereas none did in the SN. In conclusion, the temporal HF (mainly subiculum and adjacent CA1) appears to receive the main DA afferents from both VTA cells and medial half of SN, pars compacta, whereas the septal HF (particularly CA1) receives its DA input from neurons located in the ventral half and in the upper and lower borders of the VTA.

Interaction of cholinergic-dopaminergic systems in the regulation of memory storage in aversively motivated learning tasks.

These experiments examined the interaction between muscarinic cholinergic and dopaminergic systems in the modulation of memory storage. Male CD1 mice (25-30 g) were trained in an inhibitory avoidance (IA) and a Y-maze discrimination (YMD) task. The first experiment examined the dose-response effects, on retention, of agonists and antagonists specific for either D1- or D2-receptors. Immediately posttraining mice were given i.p. injections of saline, the D1-receptor agonists SKF 38393 (3.0, 10.0 or 30.0 mg/kg) or SKF 77434 (3.0, 10.0 or 30.0 mg/kg), the D1-receptor antagonist SCH 23390 (0.03, 0.1, or 1.0 mg/kg), the D2-receptor agonist quinpirole (0.3, 1.0 or 3.0 mg/kg) or the D2-receptor antagonist sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg). Retention was tested 48 h later. The drugs affecting D1-receptors did not affect retention. In contrast, in both tasks quinpirole enhanced retention and sulpiride impaired retention. In the IA task, quinpirole (3.0 mg/kg) blocked the retention impairing effects of the muscarinic cholinergic antagonist atropine (10.0 mg/kg), and sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg) significantly attenuated the memory enhancing effects of the muscarinic cholinergic agonist oxotremorine (35.0 or 70.0 micrograms/kg). D1-receptor agents did not modify the effects of either atropine or oxotremorine on retention of the IA response. These findings suggest that the effects of cholinergic muscarinic agents on retention of the IA response are mediated by influences involving D2-dopaminergic mechanisms. In the YMD task, atropine (10.0 mg/kg) blocked the memory-enhancing effects of quinpirole (3.0 mg/kg) and oxotremorine (35.0 or 70.0 micrograms/kg) attenuated the memory impairing effect of sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

The organization of nucleus tegmenti pedunculopontinus neurons projecting to basal ganglia and thalamus: a retrograde fluorescent double labeling study in the rat.

The organization of nucleus tegmenti pedunculopontinus (PPN) projections to the basal ganglia and thalamus was studied in the rat by using retrograde transport of fluorescent dyes. Fast blue was injected into the substantia nigra (SN) while Nuclear yellow was delivered to one of the following nuclei: globus pallidus (GP), entopeduncular nucleus, subthalamic nucleus (STN) or parafascicular nucleus of the thalamus. Retrogradely labeled cells were observed throughout the PPN without topographical arrangement. The cells labeled from the SN outnumbered those labeled from other structures. In all cases the majority of cells were single labeled and only a few cells double labeled from SN-GP or SN-STN were found. Labeled cells were either fusiform or multipolar in shape. These data suggest that distinct PPN cells project to their basal ganglia and thalamic targets without a prominent branched organization.

Effects of the retrorubral field stimulation on the excitability of the rat hippocampus in vivo.

We studied in vivo the influences exerted by the retrorubral field (RRF) on the on commissural-evoked CA1 pyramidal cell excitability in the rat hippocampal formation (HF). The stimulation of RRF before the activation of the contralateral CA3 area evoked in all the studied rats a reduction in amplitude of the evoked population spike in the CA1 pyramidal cell body layer of both the dorsal and the ventral HF. No significant differences in the intensity of the inhibitory effect were observed between dorsal and ventral parts of the HF. The stimulation of the ipsilateral RRF reduced the amplitude of the evoked population spike in a higher degree with respect to the contralateral side. Since these side-to-side differences were significant, it can be concluded that the RRF-induced inhibitory effect is stronger on the ipsilateral CA1 pyramidal cells. The inhibitory effect appears within 0.1 s of stimulating the RRF, reaches its maximal effect around 0.4-0.5 s following the conditioning train and returned to its control size after 5 s.

The dopaminergic mesencephalic projections to the hippocampal formation in the rat.

1. The dopaminergic mesencephalic neurons projecting to the hippocampal formation are distributed in three cell groups: A8 region in the retrorubral field, A9 region in the substantia nigra and A10 region in the ventral tegmental area. 2. Anterograde and retrograde tract-tracing techniques combined with immunohistochemical procedures indicate a topographical organization of mesencephalic dopaminergic projections towards the hippocampal formation. 3. Electrophysiological evidence suggest that dopaminergic mesencephalic neurons could have a regulatory role in suppressing hippocampal excitability. 4. The functional significance of the mesohippocampal dopaminergic system is largely unknown, although it was suggested that this projection could have a role in methamphetamine-produced hypermotility and in modulation of memory processes.

Anterograde and retrograde tracing of projections from the ventral tegmental area to the hippocampal formation in the rat.

Employing anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L), and a triple labeling protocol using retrogradely transported fluorescent tracers, we examined the projections from the ventral tegmental area (VTA-A10) to the hippocampal formation (HF) in the rat. Injections of PHA-L into VTA resulted in labeling in the ventral subiculum (stratum oriens and molecular layer) and in the adjacent CA1 field (stratum oriens, pyramidal, suprapyramidal and molecular layers) of HF. Additional labeling was observed in the stratum oriens of CA3 and in the hilus of fascia dentata. In the dorsal HF labeling was present in the subicular and CA1 field polymorphic layers. The distribution of VTA neurons projecting to the HF was also examined by injecting retrograde fluorescent tracers (Fluoro Gold, Fast Blue, and Nuclear Yellow) in several hippocampal areas. The most abundant VTA-HF projections originate from the upper and lower edges and the lower half of the VTA. These terminal fields in the HF match with the hippocampal areas projecting to the nucleus accumbens. The VTA, via projections to interconnected regions of the HF and nucleus accumbens, may modulate the hypothesized functional link between the limbic system and basal ganglia.

CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage.

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.

Angiomyofibroblastoma and aggressive angiomyxoma: two benign mesenchymal neoplasms of the female genital tract. An immunohistochemical study.

We describe a rare case of angiomyofibroblastoma (AMF) of the vulva and one case of aggressive angiomyxoma (AAM) of the pelvic region and, with the help of an extensive revision of the literature, we attempt to define their histogenesis and peculiar biological behaviour by an immunohistological evaluation. Our results indicate that AAM, which is characterized by the presence of a high content of glycosaminoglycans in the stroma, expresses uniformly vimentin and hyaluronate receptor CD44, and heterogeneously muscle specific actin (MSA) and desmin, while AMF displays a positive reaction for vimentin, desmin and laminin, and only a weak and heterogeneous positivity for CD44. Both AMF and AAM showed no immunohistochemical reactivity for alpha-smooth muscle actin (ASMA), myoglobin, cytokeratin, collagen type IV, CD68 and S-100. The stromal cells of AAM were negative for laminin. These findings support the suggestion of an origin of the two entities by a common myofibroblastic progenitor, which normally occurs in the lower female genital tract and subsequently undergoes a neoplastic transformation. The expression of CD44 by AAM, which has never been reported before, could be responsible for its more aggressive behaviour, because this receptor is able to mediate migration of neoplastic cells on a hyaluronate rich extracellular matrix. It is speculated that the neoplastic cell of the AAM and AMF of the vulva is a specific myofibroblast which probably arises from undifferentiated mesenchymal cells normally occurring in the lower female genital tract.

Consumption of a highly palatable food induces a lasting place-conditioning memory in marmoset monkeys.

Highly palatible foods may induce addiction-related behaviors. However, this has yet to be established in non-human primates. Therefore, we evaluated whether marmoset monkeys (Calllithrix penicillata) acquire a conditioned-place-preference (CPP) for chocolate and if this response is detectable after a 24-h and 15-day period. Subjects were first habituated to a two-compartment CPP box and then randomly assigned to a chocolate or control group. Thereafter, they were given access to only one compartment during daily 15-min conditionings, held on six consecutive days. On each trial, the chocolate group received pieces of chocolate (50g) in this context, whereas controls were not given a food reward. Marmosets were subsequently tested for preferring this (food) paired context after a 24-h and 15-day interval. During conditioning, individual foraging and the amount of chocolate ingested by each pair of the chocolate group remained constant. However, compared to pre-CPP levels, the time spent inside/in contact with the conditioned compartment increased significantly, while the latency to first entry decreased on both post-CPP intervals. For controls, the parameters remained unaltered. Thus, chocolate induced a persistent CPP response-an aspect usually associated with drug-related rewards.

Effects of d-amphetamine on short- and long-term memory in spontaneously hypertensive, Wistar-Kyoto and Sprague-Dawley rats.

Diverse studies indicate that the attention deficit hyperactivity disorder (ADHD) is associated with alterations in encoding processes, including working or short-term memory. Some ADHD dysfunctional domains are reflected in the spontaneously hypertensive rat (SHR). Here SHR-saline group showed significantly poor STM and LTM relative to SD and WKY saline rats. SD and WKY rats treated with d-amphetamine displayed better STM and LTM, compared to SD-vehicle, WKY-vehicle or SHR-d-amphetamine groups.

[Projections of the Tsai tegmental ventral area to the hippocampus: a study of the rat using the Fink-Heimer technic]. / Proiezioni dell'area ventrale tegmentale del Tsai all'ippocampo: uno studio eseguito sul ratto con la tecnica del Fink-Heimer.

The projection from the ventral tegmental area of Tsai (VTA-A10) to the hippocampal formation was investigated in the rat by means of the Fink-Heimer technique, after VTA destruction by electrolytic lesion or local injection of 6-hydroxydopamine (6-OHDA, 1 micrograms/0.5 microliters). Degenerated fibers are prevalently present in the ventral subiculum and CA1 field. These areas match with the area projecting towards the nucleus accumbens. Thus the dopaminergic meso-cortico-limbic pathway could modulate the HF-striatal projection which represents a link between the limbic and central motor systems.