Laura Calzà

Peptide plasticity in primary sensory neurons and spinal cord during adjuvant-induced arthritis in the rat: an immunocytochemical and in situ hybridization study.

Chronic polyarthritis due to complete Freunds adjuvant injection is characterized by severe inflammation and pain. In the present immunocytochemical and in situ hybridization study on the rat, we quantitatively investigated peptide and peptide messenger RNA expression in the sensory circuit at the spinal level, i.e. sensory neurons in the dorsal root ganglia and in nerve endings and local neurons in the dorsal horn of the spinal cord. The immunocytochemical experiments were carried out five, 13 and 21 days after complete Freunds adjuvant injection, whereas in situ hybridization study was performed after 21 days from complete Freunds adjuvant injection. The main results in the present study are the following: (i) a decrease in substance P-, calcitonin gene-related peptide- and galanin-like immunoreactivities in dorsal root ganglia is observed five days after complete Freunds adjuvant injection, with recovery (calcitonin gene-related peptide and galanin) or even an increase (substance P) after 21 days; (ii) calcitonin gene-related peptide, substance P and galanin peptide levels are increased in dorsal root ganglia after 21 days; (iii) opioid peptide (enkephalin and dynorphin), substance P and galanin messenger RNAs are strongly up-regulated in dorsal horn neurons after 21 days; (iv) neuropeptide Y content increases in dorsal root fibres and neuropeptide Y messenger RNA levels decrease in spinal neurons after 21 days; and (v) a dramatic decrease in calcitonin gene-related peptide and cholecystokinin messenger RNA levels is found in motoneurons in the ventral horn after 21 days. These data indicate that peptide expression in dorsal root ganglia and the spinal cord is markedly influenced by severe inflammation with distinct and individual temporal patterns, which are also related to the severe rearrangement of joint structure during polyarthritis. The increase in galanin levels in dorsal root ganglia 21 days after complete Freunds adjuvant injection can be related to the structural damage of nerve fibres. Thus, there may be a transition from inflammatory to neuropathic pain, which could have consequences for treatment of patients with rheumatoid arthritis.

Inhibin subunits in human placenta: Localization and messenger ribonucleic acid levels during pregnancy

This study describes the difference in distribution and levels of inhibin alpha, beta A- and beta B-subunit messenger ribonucleic acids in human placenta during pregnancy. Northern blot analysis indicated that inhibin alpha messenger ribonucleic acid is present in placental extracts collected at the early stage of gestation. Hybridization to inhibin beta A messenger ribonucleic acid was detected in the first trimester but in much lower levels. However, the intensity of the hybridization signal for inhibin alpha- and beta A-subunit messenger ribonucleic acids was greater in extracts prepared from term placentas than in those from the first or second trimester of pregnancy. Low levels of inhibin beta B-subunit messenger ribonucleic acid were observed only in extracts prepared from term placenta. At both early stage and term gestation trophoblast cells showed a positive fluorescent signal with the inhibin alpha-, beta A- and beta B-subunit-specific antisera. However, whereas inhibin alpha-subunit was localized in the cytotrophoblast, inhibin beta B-subunit immunoreactivity was observed in the syncytial layer of the villi, and inhibin beta A-subunit was widely distributed. The different distribution of immunoreactive inhibin subunits was confirmed by in situ hybridization, showing the different localizations of the inhibin messenger ribonucleic acids. These results showed that (1) human placenta produces the inhibin alpha- and beta A-subunits as early as the first trimester of pregnancy, (2) messenger ribonucleic acid levels for each of the three inhibin subunits are highest at term, and (3) immunoreactive inhibin subunits are localized differently in placental villi.

Stress promotes major changes in dopamine receptor densities within the mesoaccumbens and nigrostriatal systems

This study investigated the effects of stress on brain dopamine receptor densities in two inbred strains of mice. Analysis of [3H]SCH23390 binding by quantitative autoradiography revealed that repeated restraint stress significantly increases D1-like receptor density in the nucleus accumbens of mice of the DBA/2 strain whist reducing it in the caudate-putamen of C57BL/6 mice. No significant changes in D2-like receptor quantified by [3H](-)-sulpiride binding were observed in caudate, substantia nigra and accumbens of stressed C57BL/6 mice. Instead, in DBA/2 mice, stress significantly increased D2-like receptor density in the nucleus accumbens whilst reducing it in the substantia nigra. Finally, stress significantly increased D2-like receptor density within the ventral tegmental area of C57BL/6 mice whilst significantly reducing it in mice of the DBA/2 strain. These results indicate that stress promotes major changes in mesoaccumbens and nigrostriatal dopamine receptor densities. The direction of these changes depends on receptor subtype, brain area and strain. Moreover, the opposite changes of D2-like receptor densities promoted by stress in the ventral tegmental area of the two inbred strains of mice suggest that mesoaccumbens dopamine autoreceptors density might be controlled by a major genotype x stress interaction.

Distribution of thyrotropin-releasing hormone receptor messenger RNA in the rat brain: An in situ hybridization study

Based on the recent cloning of the mouse thyrotropin-releasing hormone receptor, oligonucleotide probes complementary to the DNA sequence were constructed and used for in situ hybridization studies on the rat brain. Thyrotropin-releasing hormone receptor messenger RNA was found in many areas of the brain, mostly showing high degree of overlap with the distribution thyrotropin-releasing hormone binding sites as previously revealed in autoradiographic studies. Thus, a strong signal was observed in the accessory olfactory bulb, the perirhinal sulcus, the ventral aspects of the hippocampal formation, some amygdaloid nuclei, the diagonal band nucleus, parts of nucleus accumbens, the bed nucleus of the stria terminalis, dorsomedial, lateral and perifornical hypothalamic regions, the septohippocampal nucleus, parts of the vestibular complex, as well as many bulbar motoneurons including the facial, dorsal vagal, ambiguus and hypoglossal nuclei, the superficial layer of the spinal trigeminal nucleus, and motoneurons and dorsal horn neurons in the spinal cord. Cells within one and the same nucleus expressed varying levels of thyrotropin releasing hormone receptor messenger RNA suggesting marked differences in rate of receptor synthesis. Most of these areas receive an input by thyrotropin-releasing hormone-positive nerve endings. Taken together these results suggest that thyrotropin-releasing hormone receptors are mostly localized in the vicinity of the cell bodies which express thyrotropin-releasing hormone receptor messenger RNA and mediate the wide range of actions that have been recorded after administration of exogenous thyrotropin-releasing hormone.

Thyroid hormone-induced plasticity in the adult rat brain.

It is well known that thyroid hormone plays a crucial role in the development and maturation of the nervous system. However, little is known about the role of thyroid hormone in the adult brain. In this short review we have dwelt on this point, with regard to the role of thyroid hormone on neuropeptide gene expression regulation in the paraventricular nucleus of the hypothalamus and in extrahypothalamic brain areas, on neurotrophin and neurotrophin receptor expression in the hippocampus and basal forebrain in basal conditions, and after neurotoxic challenges. Effects of hypothyroidism are discussed in view of a possible role of thyroid status in brain aging quality.

Response of Hypothalamic Peptide mRNAs to Thyroidectomy

Using in situ hybridization histochemistry, we have investigated the effect of thyroid hormone on the expression of several peptide mRNAs in the hypothalamic paraventricular nucleus (PVN) of adult male rats. Hypothyroidism was induced by surgical ablation of the thyroid gland. The animals (control sham-operated, thyroidectomized, thyroidectomized+T4 replaced rats) were studied 28 and 50 days after surgery. Sections of the PVN were hybridized using synthetic oligonucleotide probes complementary to mRNA for thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), galanin (GAL), enkephalin (ENK), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and vasopressin (VP). GAL mRNA was also analyzed in the anterior paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus. At the PVN level, a feedback effect of thyroid hormone on TRH synthesis was demonstrated by the TRH mRNA increase in hypothyroidism and by its decrease in hyperthyroidism. Hypothyroidism caused a dramatic decrease in GAL mRNA in parvo- and magnocellular PVN neurons both 28 and 50 days after thyroid ablation, whereas no effect was seen in VP mRNA, the main peptide hormone coexisting with GAL. The T4 replacement prevented the GAL mRNA impairment. Hypothyroidism did not influence GAL mRNA in the anterior PVN, perifornical area or in the arcuate nucleus, whereas a decrease in GAL mRNA was observed in the dorsomedial nucleus. VIP mRNA, which is undetectable in the PVN of normal animals, was present in several PVN neurons after thyroidectomy. CRH mRNA was decreased after thyroidectomy, whereas the T4 restitution caused an upregulation. The levels of ENK or NT mRNA were not significantly affected by the thyroid status. The present results show that, in addition to TRH mRNA, other hypothalamic peptide mRNAs are affected by thyroid hormone levels.

Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in arbitrary units, is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.

Thyroid Hormone Regulates NGF Content and p75LNGFRExpression in the Basal Forebrain of Adult Rats

Several lines of data from human and animal studies have suggested a role of thyroid hormone in the regulation of cholinergic neurons in the adult brain. In this study we have investigated the content of nerve growth factor (NGF) and the expression of NGF low affinity receptor (p75(LNGFR)) in the basal forebrain of adult hypothyroid rats. We describe an increase of both NGF and p75(LNGFR) expression in the basal forebrain of adult hypothyroid rats. The administration of colchicine up-regulates p75(LNGFR) expression in both hypo- and control rats, whereas it fails to down-regulate choline acetyl transferase mRNA expression during hypothyroidism. These data offer a possible neurobiological explanation to cognitive defects observed during adult hypothyroidism in humans.

NEUROTROPHINS AND THEIR RECEPTORS IN THE ADULT HYPO- AND HYPERTHYROID RAT AFTER KAINIC ACID INJECTION : AN IN SITU HYBRIDIZATION STUDY

Thyroid hormone plays a key role in trophic events during development of the central nervous system. In spite of neurological and psychiatric symptoms associated with adult hypothyroidism, the role of thyroid hormone in mature brain function is less clear. In this paper we investigated the effect of thyroid status on kainic acid‐induced up‐regulation of mRNAs for members of the nerve growth factor family and related receptors in adult male rats by means of in situ hybridization. We found that in hypothyroid rats there is a dramatic attenuation of the kainic acid‐induced up‐regulation of mRNA levels for nerve growth factor, brain‐derived neurotrophic factor and tyrosine kinase trkB in euthyroid rats. A trend to reduced c‐fos mRNA up‐regulation, which did not reach significance, was also found, whereas the increase in c‐jun mRNA after kainic acid was similar in eu‐, hypo‐ and hyperthyroid rats. These data indicate a severe impairment of the regulation of neurotrophin synthesis after excitotoxin administration in the hippocampus during adult hypothyroidism. Possible roles of thyroid hormone in molecular, biochemical and metabolic mechanisms of this defect are discussed.

Further evidence for a role of nitric oxide in experimental allergic encephalomyelitis: aminoguanidine treatment modifies its clinical evolution

The role of nitric oxide (NO) in inflammatory/demyelinating diseases is undergoing extensive investigation as a potential target for therapeutic intervention. However, interference with NO production has resulted in contrasting effects on the development of experimental allergic encephalomyelitis (EAE), the most widely used experimental model for multiple sclerosis (MS). Purpose of this paper was both the analysis of the individual clinical evolution of EAE induced in Lewis female rats by active immunisation and the evaluation of the effect of treatment with aminoguanidine, a selective inhibitor for the inducible isoform of nitric oxide synthase (iNOS). In our experimental model, relapse occurred in 66% of animals. Aminoguanidine treatment, started 3 days before immunisation, guaranteed a complete recovery from the acute phase and a delayed, milder relapse. Moreover, 79 days after immunisation inflammatory cellular infiltrates in the spinal cord were reduced. These data further support the involvement of NO in EAE evolution.