Brigada de determinación de causas

A neuronal culture, like any living system, is a complex system integrated by a myriad of different components and interactions. While Each of its components is essential for the well functioning of the system, we do not need to characterize each and every one of them to the uttermost detail to be able to understand the system as a whole. In this section we review some of the main concepts and mechanisms from physiology and biology that are needed to understand the behavior of neuronal cultures, specially when it comes to spontaneous activity in two–dimensional cultures.

1.4.1 Connectivity

Understanding the connectivity patterns between neurons is a daunting task (which we will explore inChapter 6), where each neuron in the living brain can make synapses with thousands of other neurons, sometimes making several connections with the same target. In 1986 researchers completed the full connectivity map (con- nectome) of C. elegans [White 1986], a nematode with only 302 neurons. Its con- nectivity map, consists of 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions, and although this map is invariant, i.e., the exact connections between neurons are always the same, it proved to be an extremely difficult task. Recent technological advances are making it possible to scale these studies to higher organisms, but there are still many challenges to overcome [Lichtman 2014]. Being able to obtain the full connectome from many organisms and specimens is extremely important [Sporns 2012]. In fact, from a philosophical point of view, the connectome is what gives every one of us our identity, our ’self’. In the words of S. Seung, ’we are our connectome’ [Seung 2013]. From a more practical point of view, the data obtained from connectomes is going to greatly improve our understanding of the different wiring mechanisms that give shape to the different brain functions. Even if ’we are our connectome’, every one of us is different, and yet, able to function. Unraveling the different universal rules that guide synapse formation and maintenance is the key to understanding brain function. Probably one of the most important tasks for the neuroscience community in the upcoming decades. In the same direction, several studies have appeared recently trying to untangle the universal principles behind synapse formation. Q. Wen and coworkers observed that most shapes of dendritic arbors are self–similar and can be described by an universal functional form [Wen 2009]. At the same time, several groups have found that the shapes of axonal and dendritic arbors and their connectivity patterns can be described with very few parameters [Snider 2010], and also that a very simple rule is enough to describe neuronal branching [Cuntz 2010], as well as a similar scaling

1.4. The physiology of neuronal cultures 29 law for optimal dendritic wiring [Cuntz 2012]. When it comes to particular brain areas, however, things are rather more complicated, e.g., D. Bock and coworkers found that inhibitory neurons in the cortex receive connections from the excitatory neurons with a broad range of orientations, with a lack of specificity [Bock 2011]. At the same time, K. Briggman and coworkers found highly specific connectivity patterns in the retina [Briggman 2011] regarding the computation of direction selectivity.

More specifically in neuronal cultures, Müller and coworkers [Müller 1997] char- acterized the different populations of excitatory and inhibitory neurons in hy- pothalamic cultures, and were able to estimate the ratio of inhibitory to excitatory neurons to be 2:3. They also observed that inhibitory neurons formed three times more connections than excitatory ones and also that there was a high number of excitatory–inhibitory reciprocal connections. In the laboratory of M. Segal they have also worked extensively on the physiology of neuronal cultures, and in particu- lar, they characterized how cell density determines the connectivity and morphology of dendrites and spines [Ivenshitz 2010]. They observed an inverse correlation be- tween synaptic strength and cell density, while the strength of spontaneous release remained unchanged. Their results suggest the presence of active mechanisms (plasticity) in the neuron that tune synaptic strength based on its surrounding and their activity.

Given that most neurons in cortical and hippocampal cultures are pyramidal neurons

[Spruston 2008], it might be tempting to directly extrapolate from electrophysio-

logical results in these tissues. However, it has to be done with extreme care. For example, it was observed that in layer V of the neocortex, the excitatory post– synaptic potentials (EPSPs) from pyramidal neurons show strong attenuation based on distance [Williams 2002], i.e., the amplitude of the EPSP decayed as it traveled from the synapse to the soma. On the other hand, this attenuation is not present in CA1 hippocampal pyramidal neurons [Magee 2000], where the neurons appear to have developed mechanisms of ’synaptic scaling’, so that they can correct the amplitude decay.

1.4.2 Synapse formation and plasticity

The formation and development of connections during maturation in a neuronal cul- ture is an intricate process that involves many different factors (see [Marom 2002] for a review). Neurons in culture start to develop neurites right after plating, and as soon as the cells become spontaneously active, there is a rapid increase in synapse formation, promoted by this activity. The number of total synapses, however, starts to decrease after a few weeks, although the specific time at which this process starts varies greatly between preparations of different laboratories.

30 1. Introduction

The development of synapses is strongly coupled to the activity, e.g., a reduction in activity induces neurite outgrowth, whereas an increase in activity has the op- posite effect [Van Huizen 1987]. Also, all synapses appear to be chemical, with no gap junctions present [Nakanishi 1998], although this might be related to the cell densities used in the studies. The development of connections is also coupled to other biological mechanisms, like the presence of neurotrophic factors. It has been shown that the presence of BDNF19triggers excitatory synapse formation twofold and also speeds up culture maturation [Jacobi 2009].

In the previous section we reported that the strength of the connections depends on cell density [Ivenshitz 2010], but it also changes during development. One week after plating the distribution of connection strengths is quite narrow, but it broadens after two weeks [Lin 2002]. The strength of the synapses also changes with the levels of activity, where long periods of inactivity increases neurotransmitter release and synapse size [Murthy 2001]. Not only that, but inactivity might also affect the neuron sensitivity to current input by regulating its ionic conductances [Desai 1999]. These mechanisms are related to homeostatic plasticity, which plays a very im- portant role during the development of the nervous system [Turrigiano 2004,

van Ooyen 2011] and posterior circuit refinement [Turrigiano 2011]. In a similar

direction, there has been interest in trying to induce long–lasting plasticity effects via external stimulation (usually with electrodes). The idea is to try to elicit a non–homogeneous plasticity response by a different mechanism, e.g., spike timing– dependent plasticity [Caporale 2008,Watt 2010]. The results, however, have been mixed. Although there has been several reports of activity–induced plasticity in cultures [Maeda 1998,Jimbo 1999,Shahaf 2001], in most cases it appears to be a non–lasting effect20. A thorough study by D. Wagenaar and coworkers tried to induce functional plasticity in cultures with several different protocols and were unable to observe any significant effects [Wagenaar 2006b].

1.4.3 Short–term synaptic depression

Short–term synaptic depression (STD) can also be considered as another type of synaptic plasticity, but on a much shorter time scale. The effects of STD on the activity of neuronal cultures have been reported in numerous studies. We have already shown some of them inSection 1.3.4from a modeling perspective. STD accounts for the reduction of synaptic efficacy after repetitive stimulation caused by the exhaustion of vesicle pools at the terminals (see [Zucker 2002] for a review). There are many reports of STD in cortical [Boudreau 2005] and hippocampal [Deuchars 1996,Staley 1998] slices, although with different effects

19_{Brain–Derived Neurotrophic Factor.}

1.4. The physiology of neuronal cultures 31

[Virmani 2006]. In vivo, however, it could very well be that it has a much smaller

importance [Reig 2006].

In dissociated cultures the effect of STD is clear. Different results [Maeda 1995,

Tabak 2001,Opitz 2002] already pointed in the direction that STD might play

a determinant role in burst termination and distribution. And later, M. Segal group showed unequivocally the role of STD in hippocampal dissociated cultures

[Cohen 2011] by directly modifying the dynamics of vesicle recycling.

The current model of synaptic dynamics is characterized by the interplay between three different vesicle pools that exists in the synapses. There exists a small vesicle pool, called the readily releasable pool (RRP) that is located at the synaptic bouton, almost touching (kissing) the membrane [Zhang 2009]. The RRP has very few vesicles (∼ 10 in a rat hippocampal synapse), which are ready to be released as soon as a signal (an axon potential) arrives. These vesicles are so close to the membrane that they can also spontaneously fuse REF. After these vesicles fuse with the membrane and release the neurotransmitters in the synaptic cleft, they undergo a slow recycling process which replenishes them and moves them to the recycling pool. The recycling pool is bigger (∼ 20 vesicles) and supplies new vesicles to the RRP with a time scale of a few seconds. The third vesicle pool is much bigger (∼ 200 vesicles) is called the reserve pool, and is often shared between various boutons. Its main task is to supply the recycle pool with new vesicles, however, it only becomes active after heavy stimulation and its dynamics are extremely slow (see [Rizzoli 2005] for a full review).

The combination of the timescales associated to vesicle recycling and mobiliza- tion, specially in the RRP and the recycling pool is what gives STD its char- acteristic timescales. The timescale associated to STD varies greatly from cul- ture to culture and usually has various components. Garcia-Perez and coworkers

[Garcia-Perez 2008] found that the replenishment of the RRP can have two dif-

ferent timescales, a fast one ∼ 7 s and a slow one ∼ 1 min which is switched on under heavy use. The strength of depression might also strongly depend on the number of vesicles at each pool. This variability in the STD timescales and pool sizes might explain the wide distribution of bursting activity patterns observed in cultures [Opitz 2002,Wagenaar 2006a].

1.4.4 Spontaneous activity

We have already reviewed inSection 1.3many of the different dynamical regimes of activity observed in cultures. Here we will focus on the different mechanisms that are involved in the emergence of collective spontaneous activity in cultures, specially in 2D cultures, characterized by the presence of network–wide episodes of collective activity.

32 1. Introduction 1.4.4.1 Noise sources

The collective bursts of activity observed in two–dimensional cultures have a synaptic origin, already shown by H. Robinson and coworkers in one of the first studies of network activity in cultures [Robinson 1993]. They observed that network bursts were completely suppressed after the blockage of excitatory currents by application of CNQX (an AMPA antagonist) and APV (an NMDA antagonist). Although the propagation of spontaneous firing of individual neurons is necessary to trigger bursts, the determinants of individual spontaneous firings are not so clear. Several reports show that neuronal activity is completely suppressed by the applica- tion of CNQX and APV (not only network bursts) [Cohen 2008,Serra 2010]. Many groups have also described that there are no pacemaker or endogenously active cells in hippocampal and cortical cultures [Opitz 2002,Marom 2002], although endogenously active cells are present in spinal cord cultures [Latham 2000], and this effect might depend strongly on the extracellular medium [Su 2001].

Similarly, miniature post–synaptic currents (minis) might play an important role in spontaneous activity and be one of the main sources of noise in these systems

[Otsu 2003]. Minis are caused by the spontaneous fusing of synaptic vesicles

with the membrane and the subsequent release of neurotransmitters. It has been observed that minis have a functional role in the nervous system, from maintain- ing synaptic structures [McKinney 1999] to protein synthesis [Sutton 2007] (see

[Kavalali 2015] for a review). In cultures, minis are also observed [Serra 2010],

and in the case of dense cultures their amplitudes are comparable to the ones from evoked release [Ivenshitz 2010]. Minis might also explain why single neuron activ- ity is also blocked after CNQX and APV application, since they still require free synaptic receptors to trigger any response. Their frequency, however, might be too small to trigger single neuron firing, although they could trigger dendritic spikes instead [Golding 1998,Gasparini 2004].

1.4.4.2 Synaptic currents and other effects

There are two main neurotransmitters involved in synaptic transmission in cortical and hippocampal dissociated cultures, namely glutamate for excitatory connections, and GABA for inhibitory connections (see [Hammond 2001,Hammond 2008] for more details). Glutamate acts primarily in two different receptors, AMPA and NMDA. AMPA plays a major rule in burst generation, whereas the effect of NMDA is mostly associated to burst maintenance and long–lasting plasticity effects [Cohen 2009]. NMDA–induced currents have a much smaller amplitude than AMPA ones, and although they last longer, they are also modulated by the membrane potential of the post–synaptic neuron, having almost no effect if the post– synaptic neuron is not firing (hence its importance in plasticity, as it is only active

1.5. Connectomics 33