Políticas Educativas a nivel Institucional

Current research in neuroscience devotes extraordinary quantitative attention to methodological questions. Because the brain is so complex and multifarious, almost invariably investigations must flank the question under study from several method- ological angles: anatomical, histological, electrophysiological and computational to name but a few. As research is conditioned by the scope and validity of the methods employed; we enumerate below the main methodological issues connected to our investigations.

Reliability of voltage clamp and dendritic levels of afference Voltage clamp of

electrotonically extended neurons over their whole dendritic tree is very uncertain (Williams and Mitchell, 2008). Our experiments are nevertheless relevant because voltage clamp is likely to be good at controlling the perisomatic inputs. It is precisely those inputs that have been suggested to be responsible for the ripple in the pyramidal cell layer (Ylinen et al., 1995); our holding the cell soma at the reversal of inhibi- tion is expected to make the driving force for them negligible. Thus the inward currents we observed were either truly excitatory or, rather unlikely, due to distal inhibitory afference made visible by an insufficient reach of the clamp into the farther membrane. The fast kinetics of the observed excitatory currents are somewhat surprising in view of the electrotonic filtering imposed by propagation from far-out locations. It may well be that these events emerge from contacts of Schaffer collaterals or other CA1 axons on the basal dendrites (Deuchars and Thomson, 1996; Memmesheimer, 2010).

In vivo vs. in vitro network and slicing collateral damage Slicing causes trauma

to the retained tissue: growth factors, potassium and neurotransmitters are released into the extracellular milieu and the background activity of the network ceases. Kirov et al. (1999) studied via electron microscopy the synapse density of rat hip- pocampal slices vs. perfusion-fixed tissue representative of the in vivo situation. Slices showed about 50% more spines two hours after the procedure and the number remained stable for at least 11 hours more. The stabilization of this excess seems cou- pled to return of synaptic activity to the slice one hour post-trauma. Not all spine types are affected by this process equally. The time span of slice recordings is too short for reactive synaptogenesis or sprouting to play a role. In view of these facts, we hypoth- esize that the network wiring that produces the observed SWR is almost certainly very different from the naive network before slicing. Yet, the new synaptic connec- tions that are established do follow the chemical and geometrical constraints present (that laminar boundaries are respected even by sprouting; see Frotscher et al., 1997), so as to be able to reconstitute a network that expresses oscillations very similar to SWR as observed in vivo. We thus see the network in vitro as statistically repre- sentative of the in vivo network (excluding the severed connections running through the section boundary) but do not expect it to conserve the identity of the existing synapses or their strength. To put it simply—no memories of the living animal are to be caught in the slice. As for the situation in the minislice, we ignore what are the possible additional side effects of sectioning, in particular, Schaffer collaterals. Modeling has shown that oscillations can emerge in a recurrent network from a spon- taneously firing subpopulation. If sectioning of Schaffer collaterals steadily liberates glutamate, SWRs would be facilitated by the additional excitability even in absence of CA3 synaptic input. If the widely held hypothesis that the CA3 excitatory tonic volley is necessary for the generation of CA1 ripples is to be made compatible with the minis- lice SWR a better understanding of such potential effects is needed. Another source of worry is whether the slice is thick enough to sufficiently represent the original network. The common features of the in vitro SWR (regional initiation and propagation, phar- macological sensitivity and intracellular correlates) are retained. In addition, the more compact structure of the mouse tissue with respect to the rat, where extra thick slices (1 mm vs. typical 0.4 mm) were deemed of advantage for studies of oscillations depen-

dent on CA3 recurrent axons (Wu et al., 2005a) further legitimizes the slice model employed in this work. Schaffer collaterals are rather expansive across slice sectioning planes, i.e. along the longitudinal axis in CA (Johnston and Amaral, 2004). In this light, it is perhaps not surprising that the phenomenon survives deafferentiation of CA1—the input from CA3 could have been not needed for its appearance in the first place (if it had been, there are chances that SWR would not be visible in the slice at all).

Probing the network with patched cells vs. LFP electrodes This work has relied

on combined LFP and intracellular signals to characterize collective network activity. Both are averages of neuronal activity, and both are biased and ill-specified, in dif- ferent ways. In other words, they (mostly) represent weighted averages of synaptic activity, with different sets of weights that are unknown and vary in space and with time. Of note, weights of contributions to both LFP and currents can be positive and negative, i.e. contributions can mutually cancel. This renders the inverse problem of obtaining the synaptic activity from the measured LFP or patch signal an essentially ill-posed one, with multiple formal solutions. The only way forward is to introduce additional conditions, for example demanding a laminar structure for the study of LFPs (Makarova, 2011) or establishing a voltage clamp that minimizes currents of specific sign, which was our approach.

We assigned a somewhat preponderant role to the LFP as the reference for the SWR oscillation, instead of using our quite regular, but more variably shaped, cPSCs. Besides the historical reasons (hippocampal rhythms are defined by virtue of their LFP electrographic signature), there is the fact that patched cells show cell class-specific afference patterns and even within the same class the measured currents can differ widely: thus their sampling of the network is neither universal nor predictable.

In spite of its canonical role in establishing the network oscillatory state, LFPs present a number of challenges for interpretation. CA1 being a laminated structure, the LFP varies spatially in a more predictable fashion than currents across cells or even across single cell compartments (Spruston, 2008), but its range depends on the extent of synaptic correlations (Lindén et al., 2011) and what frequency band is examined. The LFP also varies across layers along the axis from dendrite to axon and depends on possible anisotropies of the extracellular space as well as on ionic concentrations that may vary with behavioral state. Finally, it may capture far-field effects and it is certainly influenced by overlapping spikes; precisely the ones that are customarily referenced to it in unit-to-network oscillatory synchronization studies. Notwithstanding these defects of LFPs as a tool to characterize network activity, they have been successfully employed in vitro to identify coactive cell groups during SWRs: Reichinnek et al. (2010) clustered SWRs employing a machine learning approach and established stable categories with well-defined associated sets of coactivated cells. Currents provide a less geometrical and rather more topological portrait of network activity, i.e. they emphasize connectivity onto the patched cell vs. spatial adjacency to the measuring electrode. Weighted averages of postsynaptic currents at least fix the postsynaptic partner, which offers a considerable interpretive advantage: spikes are all converging onto a postsynaptic cell of known phenotype, although they cannot at present be individuated according to source through a “PSC sorting” procedure in the spirit of spike sorting. It is indeed highly attractive to sample the network as

it is seen by the cells that have to decide their firing on the presynaptic activity. In comparison, extracellular measurements and sophisticated analysis allow to ascribe spikes to source units, but cannot tell about their targets. Current measurements at the soma have limitations due to the poor spatial range of the voltage clamp tech- nique for extended neurons (Williams and Mitchell, 2008). This downside currently renders them unusable for quantitative work on in vivo data and conditions their utility in vitro to the availability of knowledge about the spatial afference pattern of presy- naptic neurons, a condition that is probably most likely to be met in hippocampal CA1 than anywhere else (Andersen et al., 2007).

Simultaneous intracellular voltage clamp recordings with extracellular multielec- trodes may eventually bridge the gap between these two representations of network activity by e.g. enabling spike-triggered averages of currents, shedding light on the number and type of presynaptic partners of a given cell. At the same time, this may provide insight as to exactly how much information can be gleaned about sites of afference and, eventually, presynaptic delays from the shape of the currents as mea- sured postsynaptically. Our peeling reconstruction algorithm may help realize these more sophisticated analyses, by disentangling signals that overlap considerably in time. This overlap is precisely the electrographic signature of assemblies in action, the con- cept that has guided our investigation in this Thesis. Peeling reconstruction may also be generally applied to other overlapping signals, such as voltage measurements, or Ca2+ _{fluorescence imaging. The reconstruction approach, however, requires a con-}

siderable set of assumptions that must be carefully checked in each particular domain of application. In general, we have verified that time-domain methods (steep slopes, reconstruction) have advantages over spectral methods in addressing the characteristics of these high SNR signals, especially when events have to be weighted equally regard- less of their amplitude.