Papers by Giuseppe Biagini
Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused... more Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies. followed by pharmacoresistant subjects ( 2190/patient). Using another standardized assessment index, the international dollar purchasing power parities, candidates to neurosurgery are 25% of all cases and account for $5401, whereas patients affected by pharmacoresistance are 20% of all cases and account for $3010. In Italy, it has been estimated that 16% of the total direct costs for antiepileptic drugs is on newly diagnosed epilepsy, 41% on patients with seizure remission, 58% on patients with occasional seizures, and 77% on patients with pharmacoresistant epilepsy [1]. For these reasons, pharmacoresistant TLE represents large part of costs required to the health system in order to assist patients affected by epilepsy.
We studied the synchronous cortical and thalamic activities induced by low (0.5–1mM) and high (50... more We studied the synchronous cortical and thalamic activities induced by low (0.5–1mM) and high (50–100mM) concentrations of the K+ channel blocker 4-aminopyridine (4AP) in a rat thalamocortical preparation. The presence of reciprocal thalamocortical connectivity was documented by diffusion of the fluorescent tracer Di-IC18 between the somatosensory cortex and the ventrobasal complex (VB) of the thalamus in vitro. Functional reciprocal connectivity
VGF is a propeptide of 617 amino acids expressed throughout the central and the peripheral nervou... more VGF is a propeptide of 617 amino acids expressed throughout the central and the peripheral nervous system. VGF and peptides derived from its processing have been found in dense core vesicles and are released from neuronal and neuroendocrine cells via the regulated secretory pathway. Among VGF-derived neuropeptides, TLQP-21 (VGF 556−576) has raised a huge interest and is one of most studied. TLQP-21 is a multifunctional neuropeptide involved in the control of several physiological functions, potentially including energy homeostasis, pain modulation, stress responsiveness and reproduction. Although little information is available about its receptor and the intracellular mechanisms mediating its biological effects, recent reports suggest that TLQP-21 may bind to the complement receptors C3aR1 and/or gC1qR. The first aim of this study was to ascertain the existence and nature of TLQP-21 binding sites in CHO cells. Secondly, we endeavored to characterize the ligand binding to these sites by using a small panel of VGF-derived peptides. And finally, we investigated the influence of TLQP-21 on selected intracellular signaling pathways. We report that CHO cells express a single class of saturable and specific binding sites for TLQP-21 with an affinity and capacity of K d = 0.55 ± 0.05 × 10 −9 M and B max = 81.7 ± 3.9 fmol/mg protein, respectively. Among the many bioactive products derived from the C-terminal region of VGF that we tested, TLQP-21 was the most potent in stimulating intracellular calcium mobilization in CHO cells; this effect is primarily due to its C-terminal fragment (HFHH-10). TLQP-21 induced rapid and transient dephosphorylation of phospholipase Cγ1 and phospholipase A2. Generation of IP 3 and diacylglycerol was crucial for TLQP-21 bioactivity. In conclusion, our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the G q-coupled receptors, and its activation first increases membrane-lipid derived second messengers which thereby induce the mobilization of Ca 2+ from the endoplasmic reticulum followed by a slower store-operated Ca 2+ entry from outside the cell.
The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the... more The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the problem of drug refractoriness. Although recognized as a standard test, it has been evaluated only recently in the attempt to characterize the putative neuronal networks involved in seizures caused by corneal stimulation. In particular, by recording from the CA1 region we previously established that the hippocampus participates to propagation of seizure activity. However, these findings were not corroborated by using markers of neuronal activation such as FosB/∆FosB antigens. In view of this discrepancy, we performed new experiments to characterize the changes in levels of phosphorylated extracellular signal-regulated kinases1/2 (p-ERK1/2), which are also used as markers of neuronal activation. To this aim, mice underwent corneal stimulation up to three different times, in three sessions separated by an interval of 3 days. To characterize a group in which seizures could be prevented by pharmacological treatment, we also considered pretreatment with the ghrelin receptor antagonist EP-80317 (330 µg/kg). Control mice were sham-treated. Video electrocorticographic (ECoG) recordings were obtained from mice belonging to each group of treatment. Animals were finally used to characterize the immunoreactivity for FosB/∆FosB and p-ERK1/2 in the hippocampus. As previously shown, FosB/∆FosB levels were highly increased throughout the hippocampus by the first induced seizure but, in spite of the progressively increased seizure severity, they were restored to control levels after the third stimulation. At variance, corneal stimulation caused a progressive increase in p-ERK1/2 immunoreactivity all over the hippocampus, especially in CA1, peaking in the third session. Predictably, EP-80317 administration reduced both duration and severity of seizures, prevented the increase in FosB/∆FosB levels in the first session, and partially counteracted the increase in p-ERK1/2 levels in the third session. The vast majority of Frontiers in Cellular Neuroscience | www.frontiersin.org
Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns... more Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psy-chomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalog-raphy showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.
Epilepsy & Behavior, 2015
Glia can influence the outcome of an epileptogenic insult by controlling the recovery of neuronal... more Glia can influence the outcome of an epileptogenic insult by controlling the recovery of neuronal networks and functions. In particular, glia may facilitate the establishment of epilepsy by impaired removal of glutamate from synapses or by releasing inflammatory cytokines and excitatory neurotransmitters, such as interleukin-1β or, respectively, glutamate, aspartate and D-serine. Opposed to these pro-excitatory/pro-epileptogenic mediators, glia can also release molecules that restrain neuronal excitability such as neurosteroids, which are potent modulators of inhibitory currents dependent on gamma-aminobutyric acid (GABA) type A receptors. In normal conditions, neurosteroids are mainly synthesized in neurons by conversion of cholesterol to pregnenolone, a step catalyzed by the cytochrome P450 cholesterol-side chain cleavage enzyme (P450scc). Following an epileptogenic insult, astrocytes transform into reactive cells and express high levels of P450scc, thus becoming major players in ...
Current Opinion in Neurology, 2010
Neurobiology of Disease, 2010
Temporal lobe epilepsy (TLE) is a chronic epileptic disorder involving the hippocampal formation.... more Temporal lobe epilepsy (TLE) is a chronic epileptic disorder involving the hippocampal formation. Details on the interactions between the hippocampus proper and parahippocampal networks during ictogenesis remain, however, unclear. In addition, recent findings have shown that epileptic limbic networks maintained in vitro are paradoxically less responsive than non-epileptic control (NEC) tissue to application of the convulsant drug 4-aminopyridine (4AP). Field potential recordings allowed us to establish here the effects of 4AP in brain slices obtained from NEC and pilocarpine-treated epileptic rats; these slices included the hippocampus and parahippocampal areas such as entorhinal and perirhinal cortices and the amygdala. First, we found that both types of tissue generate epileptiform discharges with similar electrographic characteristics. Further investigation showed that generation of robust ictal-like discharges in the epileptic rat tissue is (i) favored by decreased hippocampal output (ii) reinforced by EC-subiculum interactions and (iii) predominantly driven by amygdala networks. We propose that a functional switch to alternative synaptic routes may promote network hyperexcitability in the epileptic limbic system.
Journal of the Autonomic Nervous System, 1990
... Effects of centrally administered clonidine and neuropeptide Y on arterial blood pressure in ... more ... Effects of centrally administered clonidine and neuropeptide Y on arterial blood pressure in the rat after transient forebrain ischemia. Auteur(s) / Author(s). GRIMALDI R. ; ZINI I. ; BIAGINI G. ; TOFFANO G. ; AGNATI LF ; Affiliation(s) du ou des auteurs / Author(s) Affiliation(s). Univ. ...
Bollettino - Lega Italiana contro l'Epilessia
We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metaboli... more We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metabolite desacyl-ghrelin in rats exposed to pilocarpine-induced seizures. Pilocarpine (380 mg/kg) was administered to Sprague-Dawley rats pretreated with scopolamine, followed by saline (control group) or the tested hormone (ghrelin and desacyl-ghrelin at 1.5 mg/kg). After induction of status epilepticus (SE), seizures were quelled by injecting diazepam (20 mg/kg). Four days after SE, rats were sacrificed to evaluate lesions in hippocampus. Pilocarpine did not induce seizures in 20% and SE was prevented in 60% of desacyl-ghrelin treated rats. In ghrelin treated group, all rats developed seizures and SE. Interestingly, both ghrelin and desacyl-ghrelin prevented post-SE mortality. Hippocampal lesions were larger in control rats than in ghrelin or desacyl-ghrelin treated rats (p < 0.05 unpaired Student’s t-test). We thereby conclude that desacyl-ghrelin possesses antiepileptic properties in th...
Current Medicinal Chemistry, 2014
Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused... more Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies. followed by pharmacoresistant subjects ( 2190/patient). Using another standardized assessment index, the international dollar purchasing power parities, candidates to neurosurgery are 25% of all cases and account for $5401, whereas patients affected by pharmacoresistance are 20% of all cases and account for $3010. In Italy, it has been estimated that 16% of the total direct costs for antiepileptic drugs is on newly diagnosed epilepsy, 41% on patients with seizure remission, 58% on patients with occasional seizures, and 77% on patients with pharmacoresistant epilepsy [1]. For these reasons, pharmacoresistant TLE represents large part of costs required to the health system in order to assist patients affected by epilepsy.
Epilepsy & Behavior, 2015
Journal of Neurophysiology, 2015
Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated... more Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated to increase in the early phase of several types of brain lesion. However, their role in the pathogenesis of tissue damage is still debated. In the present paper the effects of treatments with alpha-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase, have been investigated in a model of transient forebrain ischemia. Three treatment schedules were used: alpha-difluoromethylornithine treatment was either started 3 hr before and repeated 1 hr after the insult, or started at the time of the insult and continued for 3 or 7 days after post-ischemic reperfusion. The rats were sacrificed 4 hr, 7 or 40 days after reperfusion, respectively. The acute experiment demonstrated that alpha-difluoromethylornithine can reduce the increase of glial fibrillary acid protein immunoreactivity, an early marker of astroglial reaction, in ischemic striatum. Subchronic and chronic alpha-difluoromethylornithine treatments induced a worsening of the morphological outcome of the ischemic lesion. In caudate-putamen a trend for an increase of the area of neuronal loss was present after both treatments. In the hippocampal formation, a significant increase in the severity of neuronal lesion was observed in the mildly lesioned CA3 field. In addition, other alterations of lesioned tissue were observed in alpha-difluoromethylornithine-treated animals, including increases of non-neuronal cells at 7 and especially 40 days post-lesion in striatum and CA3 hippocampal field. In conclusion, present data indicate that ornithine decarboxylase activation after ischemic lesion is a crucial factor for survival of mildly lesioned neurons and proper tissue reaction to the ischemic lesion. The experiment on acute alpha-difluoromethylornithine treatment suggests that these effects may be, at least in part, related to putrescine-induced activation of astroglial cells in the early post-lesion period.
We studied the synchronous cortical and thalamic activities induced by low (0.5–1mM) and high (50... more We studied the synchronous cortical and thalamic activities induced by low (0.5–1mM) and high (50–100mM) concentrations of the K+ channel blocker 4-aminopyridine (4AP) in a rat thalamocortical preparation. The presence of reciprocal thalamocortical connectivity was documented by diffusion of the fluorescent tracer Di-IC18 between the somatosensory cortex and the ventrobasal complex (VB) of the thalamus in vitro. Functional reciprocal connectivity
Hippocampus, 2011
The perirhinal cortex (PC), which is heavily connected with several epileptogenic regions of the ... more The perirhinal cortex (PC), which is heavily connected with several epileptogenic regions of the limbic system such as the entorhinal cortex and amygdala, is involved in the generation and spread of seizures. However, the functional alterations occurring within an epileptic PC network are unknown. Here, we analyzed this issue by using in vitro electrophysiology and immunohistochemistry in brain tissue obtained from pilocarpine-treated epileptic rats and age-matched, nonepileptic controls (NECs). Neurons recorded intracellularly from the PC deep layers in the two experimental groups had similar intrinsic and firing properties and generated spontaneous depolarizing and hyperpolarizing postsynaptic potentials with comparable duration and amplitude. However, spontaneous and stimulus-induced epileptiform discharges were seen with field potential recordings in over one-fifth of pilocarpine-treated slices but never in NEC tissue. These network events were reduced in duration by antagonizing NMDA receptors and abolished by NMDA 1 non-NMDA glutamatergic receptor antagonists. Pharmacologically isolated isolated inhibitory postsynaptic potentials had reversal potentials for the early GABA A receptormediated component that were significantly more depolarized in pilocarpine-treated cells. Experiments with a potassium-chloride cotransporter 2 antibody identified, in pilocarpine-treated PC, a significant immunostaining decrease that could not be explained by neuronal loss. However, interneurons expressing parvalbumin and neuropeptide Y were found to be decreased throughout the PC, whereas cholecystokinin-positive cells were diminished in superficial layers. These findings demonstrate synaptic hyperexcitability that is contributed by attenuated inhibition in the PC of pilocarpine-treated epileptic rats and underscore the role of PC networks in temporal lobe epilepsy. V V C 2010 Wiley-Liss, Inc.
Hippocampus, 2001
The electrophysiological properties of neurons in the rat perirhinal cortex were analyzed with in... more The electrophysiological properties of neurons in the rat perirhinal cortex were analyzed with intracellular recordings in an in vitro slice preparation. Cells included in this study (n ؍ 59) had resting membrane potential (RMP) ؍ ؊73.9 ؎ 8.5 mV (mean ؎ SD), action potential amplitude ؍ 95.5 ؎ 10.4 mV, input resistance ؍ 36.1 ؎v 15.7 M⍀, and time constant ؍ 13.9 ؎ 3.4 ms. When filled with neurobiotin (n ؍ 27) they displayed a pyramidal shape with an apical dendrite and extensive basal dendritic tree. Injection of intracellular current pulses revealed: 1) a tetrodotoxin (TTX, 1 M)-sensitive, inward rectification in the depolarizing direction (n ؍ 6), and 2) a time-and voltage-dependent hyperpolarizing sag that was blocked by extracellular Cs ؉ (3 mM, n ؍ 5) application. Prolonged (up to 3 s) depolarizing pulses made perirhinal cells discharge regular firing of fast action potentials that diminished over time in frequency and reached a steady level (i.e., adapted). Repetitive firing was followed by an afterhyperpolarization that was decreased, along with firing adaptation, by the Ca 2؉ -channel blocker Co 2؉ (2 mM, n ؍ 6). Action potential broadening became evident during repetitive firing. This behavior, which was more pronounced when larger pulses of depolarizing current were injected (and thus when repetitive firing attained higher rates), was markedly decreased by Co 2؉ application. Subthreshold membrane oscillations at 5-12 Hz became apparent when cells were depolarized by 10 -20 mV from RMP, and action potential clusters appeared with further depolarization. Application of glutamatergic and GABA A receptor antagonists (n ؍ 4), Co 2؉ (n ؍ 6), or Cs ؉ (n ؍ 5) did not prevent the occurrence of these oscillations that were abolished by TTX (n ؍ 6). Our results show that pyramidal-like neurons in the perirhinal cortex are regular firing cells with electrophysiological features resembling those of other cortical pyramidal elements. The ability to generate subthreshold membrane oscillations may play a role in synaptic plasticity and thus in the mnemonic processes typical of this limbic structure. Hippocampus 2001;11:662-672.
Pharmacology Biochemistry and Behavior, 2002
Prenatal stress strongly affects sexual dimorphism of male rats. Much less information is instead... more Prenatal stress strongly affects sexual dimorphism of male rats. Much less information is instead available on the effects of postnatal stress on sexual maturation during the so-called stress hyporesponsive period (SHRP). For this reason, we compared corticosterone-treated (CS; 10 mg/kg sc, suspended in sesame oil) or maternally separated pups (MS; 5 h/day in the first week of life) with control rats. Control and MS pups also received sesame oil injections. The effects of these procedures on physical development (body weight and eye opening), sexual maturation [anogenital distance, testis weight, 3beta-hydroxysteroid dehydrogenase/(Delta5-4) (3betaHSD) isomerase activity and time to testis descent] and glucocorticoid receptor (GR) immunoreactivity in the testis were examined. Corticosterone treatment significantly (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.05) advanced testis descent and increased testis weight and 3betaHSD activity at puberty. In addition, adult CS rats presented higher levels of GR immunoreactivity in testicular tubules when compared to control and MS rats. No differences were found between control and MS rats. On this basis, we propose that the silencing of adrenocortical function during the SHRP could be finalized to preserve sexual maturation from the influence of glucocorticoid effects. As SHRP is unique to rodents, this phenomenon could be related to their successful reproductive strategy.
Nutrition, Metabolism and Cardiovascular Diseases, 2003
It is demonstrated that dietary habits play a role in cardiovascular diseases. In stroke-prone sp... more It is demonstrated that dietary habits play a role in cardiovascular diseases. In stroke-prone spontaneously hypertensive rats (SHRsp), concomitant salt loading and a Japanese-style diet greatly accelerate hypertension and the appearance of cerebrovascular lesions by directly damaging arterial vessels. A number of studies have characterised medium and small vessel lesions in SHRsp, but little attention has been paid to the changes in the wall structure of large arteries induced by exposure to a salt-enriched diet. The aim of this study was to investigate the effects of a Japanese-style diet and salt loading on the thoracic aorta. Two-month-old SHRsp were kept on a Japanese-style diet with 1% sodium chloride solution replacing tap water. Two months later, they were sacrificed and compared with age-matched or two-month-old control SHRsp kept on a standard diet and tap water in terms of the histomorphometry, ultrastructure and biochemical composition of the thoracic aorta. The vessel was consistently thicker in the four-month-old SHRsp (+20%, p &lt; 0.05 vs two-month-old rats) regardless of diet. The salt-loaded SHRsp showed a significant reduction in elastic fibre density (-20%, p &lt; 0.05 vs two-month-old rats) and an increase in the other matrix components (%), whereas the four-month-old controls showed preserved elastic fibres and a significant increase in the other matrix components (+65%, p &lt; 0.05 vs two-month-old rats). There was a considerable increase in the amounts of 4-OH-proline (+147%), 5-OH-lysine (+174%) and desmosines (+360%) in the four-month-old controls vs their two-month-old counterparts (p &lt; 0.01), but not in the salt-loaded animals. Ultrastructural analysis revealed clear damage and accelerated aging in the thoracic aorta of the salt-loaded SHRsp. Salt loading and a Japanese-style diet destabilize thoracic aorta architecture in SHRsp after two months of treatment.
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Papers by Giuseppe Biagini