Augusto Innocenti

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula - I. Endogenous control by the circadian system

The daily pattern of locomotor activity of the ruin lizard Podarcis sicula in its natural environment changes from unimodal in spring (with only one activity peak per day) to bimodal in summer (with two well-separated activity peaks per day) and it becomes unimodal again in autumn. In order to establish whether such seasonal changes in pattern might be at least in part controlled by endogenous temporal programs, lizards were collected at different times of the year and immediately after capture their locomotor behavior was tested in the laboratory under constant temperature (29°C) and in darkness. For some individuals tested in the laboratory the locomotor pattern previously expressed in the field was known. Seasonal differences in pattern have been unequivocally found to have an endogenous component, as most lizards in constant conditions retained the locomotor pattern shown in the field during the same season. Besides, in the bimodal lizards the freerunning period of locomotor rhythms (τ) was significantly shorter and circadian activity time (a) longer than in the unimodal ones. Altogether the data are compatible with the idea that both the interdependent changes of τ and a and the changes in locomotor pattern occurring seasonally in the circadian activity rhythms of P. sicula would depend on changes in the phase relationship between mutually coupled oscillators which drive these rhythms.

The pineal and circadian rhythms of temperature selection and locomotion in lizards

The existence of a circadian rhythm of behavioral temperature selection has been demonstrated in lizards (Podarcis sicula) held on a thermal gradient in constant darkness. This rhythm becomes temporarily abolished during 1 week following parietalectomy and 2-3 weeks following pinealectomy. Parietalectomy does not affect the locomotor rhythm, while pinealectomy invariably lengthens the freerunning period of this rhythm. These results support the contention of separate control systems for the temperature selection rhythm and the locomotor rhythm. As neither rhythm is definitively abolished by parietalectomy and pinealectomy, other pacemaking components exist elsewhere in the circadian system of Podarcis sicula which can control both rhythms.

Seasonal changes of locomotor activity patterns in ruin lizards Podarcis sicula. II: Involvement of the pineal

Seasonal differences in the locomotor activity pattern of Podarcis sicula held in constant conditions in the laboratory were found to be associated with systematic differences in both the freerunning period of locomotor rhythms (τ) and the circadian activity time (α). In order to establish whether the pineal played a role in the control of seasonal changes in circadian parameters, we examined the effects of pinealectomy in constant conditions on the locomotor behavior of lizards displaying the bimodal activity pattern typical of summer. In most lizards pinealectomy lengthened τ, shortened α and abolished the bimodal activity pattern. Pinealectomy induced a sudden transition from the typical locomotor behavior of summer, characterized by a marked bimodal pattern, short τ and long α, to the typical locomotor behavior of autumn, characterized by a unimodal pattern, a long τ and short α. These results demonstrate that the pineal plays a central role in the seasonal reorganization of the circadian system that occurs in P. sicula.

Circadian organization in the ruin lizard Podarcis sicula: the role of the suprachiasmatic nuclei of the hypothalamus

The effects of electrolytic lesions to the suprachiasmatic nuclei of the hypothalamus (SCN) on circadian rhythms of locomotor activity were examined in ruin lizards Podarcis sicula maintained in constant darkness and constant temperature (29°C). All lizards (N=15) in which the lesion damaged 80% or more of the SCN became behaviorally arrhythmic. On the contrary, locomotor rhythms persisted in all cases (N=11) when the SCN remained intact and lesions were confined to neighbouring regions of the preoptic area. Taken together with previous work which demonstrates that the pineal and the retinae are not essential for the persistence of circadian locomotor rhythmicity in Podarcis sicula and with recent evidence showing the homology between the SCN of lizards and those of mammals the present results strongly support the view that the SCN of Podarcis sicula contain the primary pacemaker(s) for locomotor rhythms.

Melatonin: A coupling device between oscillators in the circadian system of the ruin lizard Podarcis sicula

Abstract 1. 1. Chronic administration of melatonin (in silastic capsules) lengthened the free-running period of the locomotor rhythm and shortened the circadian activity time in Podarcis sicula held in constant temperature and darkness. 2. 2. Lizards displaying a bimodal pattern of activity invariably became unimodal after melatonin administration. 3. 3. The results support the hypothesis that melatonin acts as a coupling device between circadian oscillators driving the locomotor rhythm in Podarcis sicula.

Identification of circadian brain photoreceptors mediating photic entrainment of behavioural rhythms in lizards

We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti‐cone‐opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone‐opsins. We named the cloned cone‐opsin Ps‐RH2. To examine whether brain cone‐opsins mediate photic entrainment of circadian locomotor rhythms, we performed post‐transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone‐opsin Ps‐RH2 mRNA in the third cerebral ventricle of pinealectomized–retinectomized lizards previously entrained to a light–dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized–retinectomized lizards to the LD cycle for 6–9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone‐opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone‐opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.

Electrolytic lesions to the optic chiasm affect circadian locomotor rhythms in lizards

Electrolytic lesions to 85-95% of both optic nerves at the level of the optic chiasm (OC-X) induce a significant shortening of the free-running period (tau) of locomotor rhythms in Podarcis sicula held in constant temperature (29 degrees C) and constant darkness. Together with previous data in P. sicula, showing that retinalectomy (RET-X) in constant darkness also mostly induces a shortening of tau, the present results demonstrate that the retinae play a central role in the control of behavioural circadian rhythmicity independently of light perception. The fact that OC-X and RET-X affect locomotor rhythms in the same way (mostly by shortening tau), strongly supports the contention that the influence of the retinae on the circadian system is neurally mediated.

The role of extraretinal photoreception in the circadian system of the ruin lizard Podarcis Sicula

Abstract 1. 1. Combination of parietalectomy, pinealectomy and retinalectomy in the same individual does not prevent Podarcis sicula from obeying Aschoffs rule for diurnal animals. 2. 2. In these operated lizards an increase in intensity of constant illumination (LL) shortens the freerunning period of locomotor rhythms, while a decrease in LL intensity has the opposite effect. 3. 3. These results demonstrate that: (i) extraretinal photoreceptors play a role in mediating parametric effects of light on the circadian system (Aschoffs rule); (ii) extraretinal photoreceptors mediating parametric effects of light lie outside the parietal eye and the pineal.

Pineal transplantation to the brain of pinealectomized lizards: Effects on circadian rhythms of locomotor activity

Pinealectomized lizards (Podarcis sicula) whose locomotor rhythms were recorded in constant temperature (29 degrees C) and darkness were subdivided into 2 groups of hosts: Each belonging to the 1st group (experimentals) received from a donor a pineal gland, and each belonging to the 2nd one (controls) received a piece of cerebellum. Pineal transplantation induced drastic changes in the free-running period (tau) of locomotor rhythms, which were significantly greater than the tau changes induced by cerebellum transplantation. Either application or removal of melatonin implants left the locomotor rhythms of the controls completely undisturbed, showing that in absence of melatonin rhythms (pinealectomy alone abolishes blood-borne melatonin rhythms) melatonin implants are ineffective. Melatonin rhythms, however, had to be present in the experimentals, because the circadian system reacts to melatonin implants by changing tau (as if melatonin rhythms had been suppressed) and to removal of the implants by again changing tau (as if melatonin rhythms had been restored).

The Role of Nutrients in a Dietary Intervention in Improving Blood Cholesterol Profile and Lowering Cardiovascular Risk

Low-density lipoprotein cholesterol has been positively associated to an increase of cardiovascular risk by a large number of epidemiological studies. On the contrary, high-density lipoprotein cholesterol results inversely related to cardiovascular risk. In this contest plasma total cholesterol and low-density lipoprotein cholesterol concentration, and the total cholesterol - high-density lipoprotein cholesterol ratio seems to be able to predict cardiovascular risk. Diet and its composition affects various plasma cholesterol concentration and their ratios. Particularly, a reduction of saturated fat acids dietary intake is strictly related to both improve of blood lipid profile and reduction of cardiovascular events incidence. On the other hand, the evidences from epidemiologic and clinical studies are consistent in finding that the reduction of cardiovascular risk depends by nutrients used for replacing saturated fat acids. Specifically it has been demonstrated that replacement of saturated fat acids with unsaturated fat acids, either monounsaturated or polyunsaturated ones, is effective in improving cardiovascular risk. On the contrary, saturated fat acids replacement with high glycemic index refined carbohydrate actually increases cardiovascular risk. Despite that, many dietary guidelines do not give any tips about nutrients to use in replacement of saturated fat acids, underestimating the significance. In this perspective Mediterranean diet, represents an attractive dietary pattern for the improvement of blood lipid profile and the reduction of the cardiovascular risk.