Le sinapsi a nastro Dr. Paola Perin Dipartimento di Scienze Fisiologiche-Farmacologiche Cellulari-Molecolari – Università di Pavia

Localizzazione delle sinapsi a nastro Cellule ciliate Fotorecettori Cellule bipolari Altre sinapsi a nastro si trovano anche nei pinealociti e negli elettrocettori dei pesci Cellule con rilascio tonico di neurotrasmettitore (glutamato) Non generano pda: potenziali < 1 mV modulano il release 100 volte + sensibili a DV delle sinapsi convenzionali

Corpi e nastri sinaptici Cellula ciliata, sacculo di rana Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Cellule ciliate vestibolari di mammifero Cono, retina di mammifero Cellula bipolare, retina di pesce

Freeze-fracture della zona attiva Cellula ciliata Le vescicole vengono rilasciate in zone attive definite La disposizione delle zone attive e’ diversa Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Giunzione neuromuscolare

Rilascio nelle sinapsi a nastro Per I fotorecettori il problema principale e’ evitare la depressione sinaptica Per le cellule ciliate e’ la sincronizzazione temporale e la compresenza di rilascio basale ed evocato Per le cellule bipolari e’ la risposta fasica Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Rilascio nelle sinapsi a nastro – cellule bipolari Le cellule bipolari ricevono un segnale che puo’ essere tonico ma danno un segnale fasico Attivazione forte VOC: RRP con tasso di 300–500 Hz. Attivazione debole VOC: rilascio del 20% del RRP e poi rallentamento fino a 10 volte. Depressione sinaptica dovuta alla presenza di vescicole con diverse sensibilita’ per il Ca (possibilmente per diversa localizzazione) Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Rilascio nelle sinapsi a nastro – cellule ciliate Cellule ciliate acustiche Devono trasmettere un segnale molto rapido Il rilascio di trasmettitore segue l’andamento temporale degli stimoli fino a circa 1 kHz Per suoni mantenuti nel tempo, si ha depressione sinaptica Cellule ciliate vestibolari Rapidita’ del segnale meno importante Parziale attivazione del rilascio a riposo (NON massimale) Non si osserva depressione sinaptica almeno per alcuni secondi di stimolazione massimale Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Rilascio nelle sinapsi a nastro - fotorecettori Possono rimanere in condizioni di massima depolarizzazione per tempi molto lunghi (soprattutto I coni) Le sinapsi dei fotorecettori devono essere in grado di rilasciare glutamato massimalmente per periodi prolungati La velocita’ di rilascio non e’ un problema (la fototrasduzione e’ lenta) Endocitosi (almeno nei coni) avviene per vescicole, non come “bulk” Le vescicole endocitate sono subito disponibili ad un nuovo ciclo di rilascio Endocitosi puo’ venire regolata dal Ca2+ nel terminale e dal GABA rilasciato dalle cellule orizzontali Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

variazioni di capacita’ Misure di rilascio variazioni di capacita’ VOCC vescicola fusione endocitosi Capacita’ di un condensatore piano: C=k x A Alla esocitosi le vescicole si fondono con la membrana plasmatica E’ possibile rilevare le variazioni di capacita’ derivanti dalla fusione esocitotica 1 vescicola: 20-50 aF Risoluzione del metodo: 5-10 fF Si riescono a risolvere 100-500 vescicole Le cellule ciliate rilasciano fino a 10000 vescicole al secondo DCm Cm

Misure di rilascio - coloranti stirenici FM1-43 e derivati sono fluorescenti in ambiente lipidico ma si ripartiscono bene in ambiente acquoso Incubando le cellule con questo colorante le vescicole sinaptiche attivamente rilasciate si colorano Attivando l’esocitosi con le vescicole marcate il colorante viene perso (bleaching) TIRF: consente di veder eventi in prossimita’ della membrana (<100nm) La maggior parte delle vescicole vengono rilasciate in corrispondenza dei corpi sinaptici Alcune vescicole pero’ si fondono fuori dalle zone attive preferenziali Terminale di cellula bipolare Verde: vescicole Rosso:membrana Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Vescicole rilasciabili – cellula bipolare Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Terminale contiene 100000-200000 vescicole 55 sinapsi/terminale

Vescicole rilasciabili – cellule ciliate Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. 1 cellula ciliata: 10-30 sinapsi nel soma 1 sinapsi: fino a 2500 vescicole Rilascio massimale: fino a 500 vescicole/s

Elementi di una sinapsi convenzionale Sistema di riempimento vescicolare (trasportatore di neurotrasmettitore, H+-ATPasi) Sistema di aggancio delle vescicole al citoscheletro sensibile al Ca2+ (sinapsina?) Fonte di Ca2+ (canali voltaggio-dipendenti N e P/Q) Sistema di sincronizzazione del rilascio (sinaptotagmina?) Sistema di fusione (complesso SNARE) Sistema di recupero (eterogeneo) Elementi strutturali (piccolo, bassoon, etc) Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Elementi di una sinapsi a nastro Sistema di riempimento vescicolare (trasportatore di neurotrasmettitore, H+-ATPasi) Sistema di aggancio delle vescicole al citoscheletro sensibile al Ca2+ (RIBEYE, ?) Fonte di Ca2+ (canali voltaggio-dipendenti L) Sistema di sincronizzazione del rilascio (sinaptotagmina?) Sistema di fusione (complesso SNARE) Sistema di recupero (eterogeneo) Elementi strutturali (piccolo, bassoon, etc) Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Struttura molecolare di una sinapsi a nastro Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Le molecole strutturali piccolo e bassoon servono ad ancorare il corpo sinaptico alla membrana

Struttura molecolare di una sinapsi a nastro Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Il corpo sinaptico e’ composto principalmente dalla proteina RIBEYE I filamenti che collegano le vescicole non sono stati identificati A livello del corpo sinaptico e’ stata osservata la miosina V: trasporto attivo delle vescicole?

Fonti presinaptiche di Ca2+ Canali di tipo T Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Canali di tipo T Canali di tipo L Cellula bipolare Cellula ciliata interna

Fonti presinaptiche addizionali di Ca2+ Il rilascio e’ inibito dalla nimodipina (antagonista dei canali L) Il rilascio e’ inibito anche dalla rianodina (antagonista di alcuni depositi intracellulari) Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Cellula ciliata vestibolare di rana

Fonti presinaptiche addizionali di Ca2+ 50 ms @ -20 mV Depolarizzazioni brevi danno aumenti di Ca2+ transienti 500 ms @ -20 mV Depolarizzazioni prolungate danno il via a processi di amplificazione del Ca2+ (CICR) sensibili alla rianodina

Fonti presinaptiche addizionali di Ca2+ Nei coni, parte del Ca presinaptico entra attraverso canali CNG localizzati al terminale Nelle cellule bipolari, stimoli deboli attivano I canali T, stimoli piu’ forti sia I T che gli L Diversa cinetica di rilascio nelle due condizioni di stimolazione Nelle cellule ciliate vestibolari, stimoli prolungati evocano un meccanismo di rilascio da stores intracellulari Contrasta inattivazione delle correnti di Ca, consente rilascio prolungato Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Canali del Ca2+ di tipo L Cav1.1: canale del muscolo scheletrico Cav1.2: canale del muscolo cardiaco, vascolatura, neuronale Cav1.3: canale delle cellule ciliate (anche nei coni?) Cav1.4: canale dei bastoncelli (anche delle cellule bipolari?), sistema immunitario Sindromi e knock-out Knock-out per Cav1.1: completa paralisi muscolare (letale subito dopo la nascita, mdg) Knock-out per Cav1.2: letale in utero Knock-out per Cav1.3: sordita’ completa, problemi cardiaci Sindrome da cecita’ notturna legata all’X: mutazione in Cav1.4 Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Canali Cav1.3 Voltaggio-dipendenza (triangoli: canali P/Q) Cinetica di attivazione m2 fit Activation t Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Cinetica di inattivazione

Canali Cav1.4 Voltaggio-dipendenza Cinetica di inattivazione Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Attivati lentamente, ma non hanno inattivazione Ca-dipendente

Plasticita’ dei corpi sinaptici Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers. Nei fotorecettori la forma e le dimensioni dei corpi sinaptici variano durante il ciclo giorno-notte Nelle cellule ciliate vestibolari I corpi sinaptici si modificano in condizioni di alterata gravita’

Pool vescicolari nelle sinapsi a nastro RRP: vescicole rilasciate rapidamente dopo depolarizzazione SRP: vescicole rilasciate con un ritardo RRP corrisponde al numero di vescicole attaccate ai corpi sinaptici (6000 nel terminale bipolare, 300 nelle cellule ciliate acustiche)

Pool vescicolari nelle sinapsi a nastro Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.

Pool vescicolari nelle sinapsi a nastro Hair cells express a large number of Ca buffer proteins, which have the role of confining Ca increases to a very small domain. This is a presumed Ca channels cluster from the frog saccule, where Ca regulation has been studied in most detail. At the presynaptic active zones, Ca channels are thought to be clustered together with BK-type Ca-dependent K channels. (Black dots are Ca channels, white dots are BK channels). Simulations show that the presence of a large concentration of mobile buffer allows the generation of a very steep Ca gradient at the edge of the cluster, and a few hundred nm from the cluster there is virtually no Ca increase. It is interesting to note that different hair cells even within the same organ express different Ca buffers.