Massimo Zanni

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.

Increase of neuropeptide Y-like immunoreactivity in the paraventricular nucleus of fasting rats

The paraventricular nucleus (PVH) of the hypothalamus is a key region for the control of food intake. It presents a very high neuropeptide Y (NPY)-like positive innervation. In this paper we have studied the modifications of NPY-positive innervation in the PVH of 72 h starved rats vs control rats by means of semiquantitative immunocytochemistry. We observed a significant increase of NPY-like immunoreactivity in fasting rats. This result suggests a physiological role of NPY in the food intake regulation at the PVH level.

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.

Regulation of VIP mRNA expression by thyroid hormone in different brain areas of adult rat

A role of thyroid hormone in the regulation of neuropeptide synthesis has been demonstrated in different tissues. In this paper we investigated the vasoactive intestinal peptide (VIP) mRNA expression by means of in situ hybridization in several brain areas of hypo- and hyperthyroid adult rats. Neither hypo- nor hyperthyroidism modified the VIP mRNA levels in the thalamus and in the hypothalamic suprachiasmatic nucleus. In contrast, in the anterior cingulate and frontoparietal motor cortex of hypothyroid rats there was a marked increase in the signal for VIP mRNA per cell, but the number of VIP expressing neurons did not change. These data indicate that also central VIP synthesis can be influenced by the levels of circulating thyroid hormone, but that this effect is confined to specific areas and cell populations of the brain.

Iodinated-NPY binding sites: Autoradiographic study in the rat brain

Several authors have described the presence of iodinated neuropeptide-Y binding sites on membranes of the mammalian CNS. In the present study we show a mapping of iodinated-NPY binding sites in the rat brain using receptor autoradiography. The sections were incubated with 125I-Bolton-Hunter coupled NPY (0.5-03 nM), in the absence or presence of 1 microM cold NPY. Some autoradiograms are studied by means of an image analyzer (VDC 501 Tesak) equipped with the host computer PDP 11 Digital, in order to enhance the contrast of the labeling. A very high density of NPY receptors is present in the limbic regions (hippocampus, amygdaloid complex, septal nuclei), in the cortex, and in some thalamic nuclei, while in some hypothalamic regions (paraventricular nucleus and median eminence) we detected a lower amount of NPY receptors. At the mesencephalic level, the substantia nigra presents a very high density of NPY receptors.

Dopamine receptors in the striatum of rats exposed to repeated restraint stress and alprazolam treatment

Stress-related behaviors are accompanied by modification of a large number of neurotransmitters in the brain. Moreover, the binding to GABA(A) receptors does not account for all the effects of benzodiazepines. In this study we investigated the effect of repeated restraint stress and alprazolam treatment (1 mg/day os) on dopamine receptors (Bmax and Kd) in the striatum of adult rats by means of quantitative receptor autoradiography. After chronic restraint stress dopamine D1 receptors (Bmax value) decreased in the accumbens nucleus, whereas dopamine D2 receptors were not modified in any investigated area. After alprazolam treatment, a considerable increase in both dopamine D1 and D2 receptors in the striatum was observed. Chronic immobilization stress together with alprazolam treatment re-established dopamine D1 receptor density to control values in the accumbens nucleus and olfactory tubercle, whereas it resulted in an increase in dopamine D2 receptors comparable to that elicited by alprazolam treatment alone.

DA1 and DA2 receptor regulation in the striatum of young and old rats after peripheral vestibular lesion

Anatomical, lesion and functional studies have indicated that the mesostriatal dopaminergic (DAergic) system may serve as supravestibular center in posture and locomotion control. Nevertheless, no data are available on the involvement of DAergic systems during vestibular compensation. This study was designed for the analysis of DA1 and DA2 receptors in the striatum by means of quantitative receptor autoradiography 28 days after unilateral or bilateral lesion of the labyrinth in 3-month-old rats. Considering the severe decline of DA content and receptors in striatum and the difference in behavioral recovery after vestibular lesions in old age, we also analyzed 24-month-old, lesioned and unlesioned rats. In young rats, hemilabyrinthectomy caused a bilateral increase (20-30%) of DA1 receptors and a two-fold increase of DA2 receptors. In old-rats, we observed a similar modification of DA2 receptors, and a 50% increase in DA1 receptors. Bilabyrinthectomy did not modify DA1 receptor density and decreased DA2 receptor density in young animals, whereas it produced an increase in both DA1 and DA2 in old rats. This study provides evidence for the involvement of the DAergic system during vestibular compensation. Our results also indicate great biochemical plasticity of the remaining DA receptors in the striatum of old rats.

Daily Modifications of 3h-Naloxone Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study

The endogenous opioid peptides, the opiate receptors and several related behaviours, like opioid-mediated analgesia, show daily variations in different animal species including rats. The attempt to correlate the daily rhythm of opiate receptors in the central nervous system (CNS) to opiate related rhythmic phenomena requires an experimental approach with a high anatomical resolution, as the opioid distribution is very heterogeneous. In this paper we present the study of daily variations of 3H-naloxone binding sites in the different regions of the adult male rat brain, performed by means of quantitative autoradiography. Five rats are sacrificed at each investigated time of the day (0200, 0600, 1000, 1400, 1800 and 2200). The ligant is 3H-naloxone (4 nM), the quantification is performed by means of densitometric procedures (image analyzer Tesak VDC 501, computer Digital PDP 11, 3H-microscale). The statistical analysis is performed according to the single Cosinor method and the one-way analysis of variance followed by the multiple range test of Duncan. We analysed 33 different regions of the rat CNS, and the daily variations of opiate receptors are regionally selective. A circadian rhythm is found in the anterior cingulate cortex, hippocampal cortex, periventricular, medial, ventral, reticular and posterior nuclei of the thalamus, rhomboid, gelatinosus and rheuniens nuclei, lateral hypothalamus, locus coeruleus, grey substance of the pons, reticular formation of medulla oblongata, inferior olivary complex, medial part of the nucleus of the solitary tract and nucleus of the spinal tract of the trigeminal nerve. An ultradian rhythm is found in the medial and lateral preoptic areas, in the medial hypothalamus, in the medial and in the lateral nuclei of habenula. No significant variations during 24 hr according to the Cosinor analysis are found in the dorsal and lateral cerebral cortex, striatum, globus pallidus, bed nucleus of the stria terminalis, septal nuclei, lateral nucleus of the thalamus, cochlear nuclei, nucleus of the solitary tract, lateral and caudal parts, dorsal motor nucleus of the vagal nerve, XII and IX nerve nuclei. The amplitude of the daily variations observed ranges from 10 to 40%. Our results demonstrate the high anatomical selectivity of the daily modifications of 3H-naloxone binding sites in the rat CNS. They also indicate that quantitative autoradiography is a suitable and sensitive technique for these studies.

DA2/NT receptor balance in the mesostriatal and mesolimbocortical systems after chronic treatment with typical and atypical neuroleptic drugs

Typical and atypical neuroleptic drugs show several clinical and behavioral effects, possibly related to the different anatomical sites of the action in the mesolimbocortical or mesostriatal dopaminergic systems. Because of the interaction between dopamine (DA) and neurotensin (NT) in the target areas of these systems, and in order to study if the different action of typical and atypical neuroleptic drugs could be related to a modification of the DA/NT balance, we investigated DA2 and NT receptor modifications--by means of quantitative receptor autoradiography--after chronic treatment with low dosage of haloperidol, chlorpromazine, thioridazine and clozapine. We described a decrease of NT receptor density in the target areas of the mesolimbocortical system produced by all the treatments. This effect does not match with DA2 receptor modifications. On the contrary, the block of DA transmission obtained by high dosage of haloperidol induces an increase of NT receptor density. Our results further demonstrate the regulation of NT transmission by DAergic drugs.

Effect of sertraline treatment on benzodiazepine receptors in the rat brain

In this paper we describe the modification of benzodiazepine (BDZ) binding sites in the rat brain after different times of treatment with the 5-hydroxytryptamine-(5HT) uptake blocker sertraline. We investigated the effect of 8, 15 and 30 days sertraline treatment (10 mg/kg/day, i.p.) on3H-flunitrazepam binding sites. In order to describe the anatomical site of action of the drug, the experiment has been carried out by means of quantitative receptor autoradiography. After 8 days of sertraline treatment, an increase of BDZ receptor density is found in the olfactory tubercle. This effect is reversed at 15 and 30 days. At 15 days of treatment, an increase is found in the anterior cingulate cortex. This increase is still present after 30 days of treatment. At 30 days of treatment, we also found an increase of BDZ receptor density in the frontoparietal motor cortex and in the septal nuclei. The Scatchard plots obtained from the saturation experiments indicate that this increase of the receptor density is due to an increase of both the receptor number and affinity. All the other investigated areas are unaffected by the sertraline treatment. The possible neurochemical basis of these BDZ receptor regulation by sertraline and its influence in the therapeutical profile are discussed.