Neurobiology and Integrated Physiology Laboratory

Neurobiologia e Fisiologia Integrata

Referent: Prof. Paola Rossi

Co-workers: Elisa Roda, Daniela Ratto, Francesca Talpo, Maria Teresa Venuti, Martino Ramieri

The Laboratory of Neurobiology and Integrated Physiology uses electrophysiological techniques for the in vitro, ex vivo and in vivo study of Central Nervous System preparations, behavioral tests on a preclinical model and immunocytochemical and immunohistochemical techniques. A cell culture laboratory is also active.

(i) Actions of oxytocin in the central nervous system

Oxytocin, a hormone synthesized at the hypothalamic level, acts both peripherally and centrally by regulating various behavioral functions. A correlation between the oxitoninergic system and autism has recently been hypothesized. The oxytocin receptors are widely distributed in the hippocampus, and several electrophysiological studies have been performed on rodents to identify the oxytocinergic hippocampal circuits and to understand the modulatory mechanisms exerted by the neuropeptide in this area. It emerged that oxytocin facilitates inhibitory transmission in the CA1 area of the hippocampus, increasing the discharge rate of a class of GABAergic interneurons synaptically connected to pyramidal cells. Nevertheless, the mechanism by which oxytocin increases the firing rate of inhibitory neurons has not been identified yet, nor is it known how this increase affects the excitability of pyramidal neurons. Furthermore, the mechanisms through which oxytocin is able to improve the symptoms of autistic disease are not known. Our study is focused on the characterization of neuromodulation operated by oxytocin on the mouse hippocampus, trying to fill the gaps in current knowledge.

Referents: M. Toselli, Prof. G. Biella

Collaborations: 

  • B. Chini, Istituto di Neuroscienze – CNR, Milano;
  • M. Parenti, Dip. Medicina Sperimentale Università di Milano Bicocca, Milano.

 

(ii) Analysis of synaptic activity in animal models of Huntington's disease (HD)

HD is a neurodegenerative disorder caused by an autosomal dominant mutation in the IT-15 gene that codes for the huntingtin protein (Htt). The role of Htt is still unknown. The protein is ubiquitous, essential for embryogenesis, neuronal development and survival, and is also involved in synaptic activity. Expansion of the CAG triplet in exon 1 of the IT-15 gene (> 36 repeats) produces a mutated form of Htt (mHtt) which is toxic to neurons and causes extensive loss of brain neurons, especially in the cortical and striatal levels. In addition to neuronal death in the last stage of the disease, mHtt causes also progressive changes in the morphology, excitability, and synaptic properties of cortical pyramidal neurons (CPNs) and medium spiny neurons (MSNs). Therefore, the first behavioral and cognitive symptoms of HD precede neuronal death rather than being a consequence of it. The decoupling of connectivity and plasticity at the level of the CPN/MSN synapses and excitotoxicity, mainly mediated by alterations of NMDA receptors, seem crucial in the pathogenesis and progression of HD. Using multidisciplinary approaches (behavioral, electrophysiological etc.), we intend to analyze the progressive decoupling of excitatory corticostriatal synapses in HD knock-in mouse models, both in the pre-symptomatic and symptomatic stages of the disease.

Referents: M. Toselli, Prof. G. Biella

 

(iii) Functional evaluation of middle spiny neurons of the striatum differentiated from embryonic stem cells and reprogrammed from fibroblasts of patients with Huntington's disease

This research project focuses on the functional characterization of a specific class of striatal neurons, the middle spiny neurons (MSN). Through a specific differentiation protocol from human embryonic stem cells (hES, H9 line), 2D or 3D cultures of MSN will be obtained. In some lines also exposed to an induced overexpression of factors such as GSX2 and EBF1 to improve yield and quality. Furthermore, given the devastating degeneration of MSNs in Huntington's disease, our interest has also turned to the electrophysiological characterization of differentiated striatal neurons starting from induced pluripotent stem cells (hiPS), derived from fibroblasts of healthy and diseased subjects. In this way, it is possible to model Huntington's disease in vitro and provide a relevant contribution to the understanding of the multiple and still unknown molecular mechanisms underlying neurodegeneration. In addition, physiological investigations will help to consolidate the cell differentiation protocol, in order to generate authentic MSNs usable in cell therapy, which to date appears to be an excellent candidate for the treatment of neurodegenerative diseases.

Referents: M. Toselli, Prof. G. Biella
Collaborations:

  • E. Cattaneo, Dip. Bioscienze, UNIMI, Milano;
  • M. Onorati , Dept. Neurobiology, Yale School of Medicine, New Haven, Connecticut, USA.

 

(iv) Mechanisms and neuromodulation of membrane excitability in neurons of the parahippocampal cortex and neurons of the perirhinal cortex

The parahippocampal cortices (PHCs) establish bidirectional synaptic interactions with the hippocampus that are of fundamental importance for the memory and spatial orientation functions of the memory system of the medial temporal lobe. This project is focused on studying the mechanisms that govern the intrinsic excitable properties of PHC neurons, their functional implications for communications between the parahippocampal region and the hippocampus, and the neuromodulatory systems that control them. In particular, we study: 1) the intrinsic membrane mechanisms that determine the specific firing properties of the neurons of the medial entorhinal cortex (mEC); 2) membrane resonance, the underlying mechanisms, and related functional implications, in neurons of the mEC and perirhinal cortex (PRC); 3) the regulatory mechanisms through which the PRC is able to operate its characteristic function of selecting the input signals directed towards the hippocampus through the ECM, through the intervention of a complex intrinsic network, which involves both the pyramidal cells of projection and numerous classes of GABAergic interneurons that generate a local inhibition on pyramidal neurons; 4) whether and how numerous physiological neuromodulatory systems, such as those that refer to acetylcholine, dopamine, noradrenaline, etc.) affect the aforementioned properties of individual neurons and local circuits.

Referents: M. Toselli, Prof. G. Biella

 

(v) Role of endothelial Ca2 + signals in neurovascular coupling

Neurovascular coupling is the mechanism by which cerebral blood flow increases or decreases in response to corresponding changes in neural activity. The most accredited hypothesis attributes exclusively to neurons and astrocytes the ability to release vasoactive mediators in response to synaptic activity. This research project is investigating for the first time the ability of the cerebral endothelium to directly detect the synaptic release of neurotransmitters. Our attention is focusing on the sensitivity of bEND.5 cells, a widely validated model for the study of the cerebral endothelium, the neurotransmitters glutamate and acetylcholine and the neuropeptide catestatin. In details, this project aims to: 1) study the mechanisms responsible for the genesis of the intracellular oscillations of Ca2+ which arise following stimulation with the three agonists mentioned above; 2) verify whether and how these Ca2+ fluctuations induce the synthesis of nitrogen monoxide (NO), the main vasodilating agent of the brain; 3) investigate whether NO released by brain endothelial cells can act not only on vascular tone, but also on synaptic activity.

Referent: Prof. F. Moccia
Collaborations:

  • T. Angelone, Università della Calabria, Cosenza;
  • E. D’Angelo, Dip. Scienze del Cervello e del Sistema Nervoso, UNIPV;
  • S. Dragoni, University College London, London;
  • G. Guerra, Università del Molise, Campobasso;
  • D. Lim, Università del Piemonte Orientale, Novara.

 

(vi) Study of the effects of oral supplementation with the medicinal mushroom Hericium erinaceus (Lion's mane) on neurogenesis and cognitive functions in wild-type mice.

Hericium (Bull.) Pers. Is a medicinal mushroom capable of modulating the immune system and improving cognitive functions in humans. Over the last 10 years numerous in vitro and in vivo studies have been conducted to investigate its effects after oral administration. On humans, in particular, the data in the scientific literature describe a neuroprotective effect on stroke and effects of partial cognitive recovery in neurodegenerative diseases, such as dementia and Alzheimer's. In a mouse model of mice pharmacologically induced to the development of Alzheimer's, dietary supplementation with H. erinaceus prevents impairment of short-term spatial memory and visual recognition memory. There are no literature data relating to wild-type animals. This line of research studies the effects of oral administration of the mycotherapy in wild-type mice. Two aspects are addressed in particular: the ability to improve some cognitive performance related to memory and the effects on neurogenesis at the hippocampal level. Patch-clamp methods in hippocampal slices and in vivo animal behavior studies are used.

Referent: Prof. P. Rossi
Collaborations: 

  • F. Brandalise, Brain Research Institute, University of Zurich;
  • A. Gregori, MycoMedica d.o.o., Kranjska Gora, Slovenia;
  • G. Orrù, Dip. Scienze Chirurgiche, Università di Cagliari;
  • M.L. Guglielminetti, M.Rodolfi ed E. Savino, Dip. della Terra e dell’Ambiente, UNIPV.

 

(vii) Type 3 diabetes: Alzheimer's 

Nutritional Neuroscience is a new field of nutrition that deals with the study of the effects of bioactive components present in food on the CNS. In the laboratory, the effect of chronic peripheral hyperglycemia (diabetes) at the CNS level is studied. Recent studies show in animal models that a low glycemic index diet exerts neuroprotective effects, on the risk / prevention and on the progression of some neurodegenerative diseases, including Alzheimer's, in which a component of metabolic origin is recognized, and therefore considered a type III diabetes (for a review, Shieh et al. 2020, doi: 10.1007 / s12035-019-01858-5). The Laboratory has long developed functional tests in vivo, on animal models, to test, after oral supplementation, the effects of substances on explicit declarative memory, a cognitive component of particular importance in the cognitive decline of Alzheimer's disease. This research project aims to study the mechanisms of action underlying the relationship between glycemia, insulin resistance and cognitive impairment.

 

(viii) Study of the membrane currents involved in the metastasis process of human glioblastoma. 

This line of research makes use of the patch-clamp technique integrated with the wound-healing assay technique and of the time lapse microscopy for the understanding of the membrane mechanisms involved in the ability to metastasize of the IV degree glioblstoma multiforme. The Neurobiology and Integrated Physiology laboratory has been characterizing the inward rectifier Kir4.1 currents and the BK currents involved in this process for years (Ratto et al. 2019; Brandalise et al. 2020). The study of these mechanisms could lead to the development of new therapies that target voltage-gated ion channels.