Tapered Fibers Combined With a Multi-Electrode Array for Optogenetics in Mouse Medial Prefrontal Cortex.

Optogenetics offers many advantages in terms of cell-type specificity, allowing to investigate functional connectivity between different brain areas at high spatial and neural population selectivity. In order to obtain simultaneous optical control and electrical readout of neural activity, devices called "optrodes" are employed. They are typically composed of a linear array of microelectrodes integrated on a slender probe shafts combined with flat-cleaved optical fibers (FF) placed above the recording sites. However, due to tissue absorption and scattering, light delivered by the FF unevenly illuminates the region of interest. This issue is of particular relevance when cellular populations are disposed along the dorso-ventral axis, such as in medial prefrontal cortex (mPFC) where cortical layers are aligned vertically. The study presented here aims at using tapered optical fibers (TFs) in combination with a 16-electrode neural probe to better access neural populations distributed along the dorso-ventral axis in the mPFC of newborn mice, restricting light delivery over a specific portion of the cortical layer of interest. Half of the TF surface is coated with a reflecting metal blocking the light to enable light delivery from one side of the probe's shaft only, with the probe base being designed to host the fiber without interfering with the wire-bonds that connect the recording sites to a printed circuit board. Monte-Carlo simulations have been implemented to define the relative TF-probe position and to identify the light intensity distribution above the recording sites. In vivo recordings indicate that simultaneous optical stimulation and electrical readout of neural activity in the mPFC benefit from the use of the engineered TF-based optrode in terms of a more uniform light distribution along the dorso-ventral axis and the possibility of restricting light delivery to a subset of electrical recording sites of interest.

Tailoring light delivery for optogenetics by modal demultiplexing in tapered optical fibers.

Optogenetic control of neural activity in deep brain regions ideally requires precise and flexible light delivery with non-invasive devices. To this end, Tapered Optical Fibers (TFs) represent a versatile tool that can deliver light over either large brain volumes or spatially confined sub-regions, while being sensibly smaller than flat-cleaved optical fibers. In this work, we report on the possibility of further extending light emission length along the taper in the range 0.4 mm-3.0 mm by increasing the numerical aperture of the TFs to NA = 0.66. We investigated the dependence between the input angle of light (θin) and the output position along the taper, finding that for θin > 10° this relationship is linear. This mode-division demultiplexing property of the taper was confirmed with a ray tracing model and characterized for 473 nm and 561 nm light in quasi-transparent solution and in brain slices, with the two wavelengths used to illuminate simultaneously two different regions of the brain using only one waveguide. The results presented in this manuscript can guide neuroscientists to design their optogenetic experiments on the base of this mode-division demultiplexing approach, providing a tool that potentially allow for dynamic targeting of regions with diverse extension, from the mouse VTA up to the macaque visual cortex.

Kainic acid-induced albumin leak across the blood-brain barrier facilitates epileptiform hyperexcitability in limbic regions.

OBJECTIVE: Systemic administration of kainic acid (KA) is a widely used procedure utilized to develop a model of temporal lobe epilepsy (TLE). Despite its ability to induce status epilepticus (SE) in vivo, KA applied to in vitro preparations induces only interictal-like activity and/or isolated ictal discharges. The possibility that extravasation of the serum protein albumin from the vascular compartment enhances KA-induced brain excitability is investigated here. METHODS: Epileptiform activity was induced by arterial perfusion of 6 µm KA in the in vitro isolated guinea pig brain preparation. Simultaneous field potential recordings were carried out bilaterally from limbic (CA1, dentate gyrus [DG], and entorhinal cortex) and extralimbic regions (piriform cortex and neocortex). Blood-brain barrier (BBB) breakdown associated with KA-induced epileptiform activity was assessed by parenchymal leakage of intravascular fluorescein-isothiocyanate albumin. Seizure-induced brain inflammation was evaluated by western blot analysis of interleukin (IL)-1ß expression in brain tissue. RESULTS: KA infusion caused synchronized activity at 15-30 Hz in limbic (but not extralimbic) cortical areas, associated with a brief, single seizure-like event. A second bolus of KA, 60 min after the induction of the first ictal event, did not further enhance excitability. Perfusion of serum albumin between the two administrations of KA enhanced epileptiform discharges and allowed a recurrent ictal event during the second KA infusion. SIGNIFICANCE: Our data show that arterial KA administration selectively alters the synchronization of limbic networks. However, KA is not sufficient to generate recurrent seizures unless serum albumin is co-perfused during KA administration. These findings suggest a role of serum albumin in facilitating acute seizure generation.

Determinants of different deep and superficial CA1 pyramidal cell dynamics during sharp-wave ripples.

Sharp-wave ripples represent a prominent synchronous activity pattern in the mammalian hippocampus during sleep and immobility. GABAergic interneuronal types are silenced or fire during these events, but the mechanism of pyramidal cell (PC) participation remains elusive. We found opposite membrane polarization of deep (closer to stratum oriens) and superficial (closer to stratum radiatum) rat CA1 PCs during sharp-wave ripples. Using sharp and multi-site recordings in combination with neurochemical profiling, we observed a predominant inhibitory drive of deep calbindin (CB)-immunonegative PCs that contrasts with a prominent depolarization of superficial CB-immunopositive PCs. Biased contribution of perisomatic GABAergic inputs, together with suppression of CA2 PCs, may explain the selection of CA1 PCs during sharp-wave ripples. A deep-superficial gradient interacted with behavioral and spatial effects to determine cell participation during sleep and awake sharp-wave ripples in freely moving rats. Thus, the firing dynamics of hippocampal PCs are exquisitely controlled at subcellular and microcircuit levels in a cell type-selective manner.

Fast Activity Evoked by Intracranial 50 Hz Electrical Stimulation as a Marker of the Epileptogenic Zone.

Epilepsy is a disease characterized by aberrant connections between brain areas. The altered activity patterns generated by epileptic networks can be analyzed with intracerebral electrodes during pre-surgical stereo-electroencephalographic (EEG) monitoring in patients candidate to epilepsy surgery. The responses to high frequency stimulation (HFS) at 50 Hz performed for diagnostic purposes during SEEG were analyzed with a new algorithm, to evaluate signal parameters that are masked to visual inspection and to define the boundaries of the epileptogenic network. The analysis was focused on 60-80 Hz activity that represented the largest frequency component evoked by HFS. The distribution of HFS-evoked fast activity across all (up to 162) recording contacts allowed to define different clusters of contacts that retrospectively correlated to the epileptogenic zone identified by the clinicians on the basis of traditional visual analysis. The study demonstrates that computer-assisted analysis of HFS-evoked activities may contribute to the definition of the epileptogenic network on intracranial recordings performed in a pre-surgical setting.

Function of inhibitory micronetworks is spared by Na+ channel-acting anticonvulsant drugs.

The mechanisms of action of many CNS drugs have been studied extensively on the level of their target proteins, but the effects of these compounds on the level of complex CNS networks that are composed of different types of excitatory and inhibitory neurons are not well understood. Many currently used anticonvulsant drugs are known to exert potent use-dependent blocking effects on voltage-gated Na(+) channels, which are thought to underlie the inhibition of pathological high-frequency firing. However, some GABAergic inhibitory neurons are capable of firing at very high rates, suggesting that these anticonvulsants should cause impaired GABAergic inhibition. We have, therefore, studied the effects of anticonvulsant drugs acting via use-dependent block of voltage-gated Na(+) channels on GABAergic inhibitory micronetworks in the rodent hippocampus. We find that firing of pyramidal neurons is reliably inhibited in a use-dependent manner by the prototypical Na(+) channel blocker carbamazepine. In contrast, a combination of intrinsic and synaptic properties renders synaptically driven firing of interneurons essentially insensitive to this anticonvulsant. In addition, a combination of voltage imaging and electrophysiological experiments reveal that GABAergic feedforward and feedback inhibition is unaffected by carbamazepine and additional commonly used Na(+) channel-acting anticonvulsants, both in control and epileptic animals. Moreover, inhibition in control and epileptic rats recruited by in vivo activity patterns was similarly unaffected. These results suggest that sparing of inhibition is an important principle underlying the powerful reduction of CNS excitability exerted by anticonvulsant drugs.

Extracellular calcium controls the expression of two different forms of ripple-like hippocampal oscillations.

Hippocampal high-frequency oscillations (HFOs) are prominent in physiological and pathological conditions. During physiological ripples (100-200 Hz), few pyramidal cells fire together coordinated by rhythmic inhibitory potentials. In the epileptic hippocampus, fast ripples (>200 Hz) reflect population spikes (PSs) from clusters of bursting cells, but HFOs in the ripple and the fast ripple range are vastly intermixed. What is the meaning of this frequency range? What determines the expression of different HFOs? Here, we used different concentrations of Ca(2+) in a physiological range (1-3 mM) to record local field potentials and single cells in hippocampal slices from normal rats. Surprisingly, we found that this sole manipulation results in the emergence of two forms of HFOs reminiscent of ripples and fast ripples recorded in vivo from normal and epileptic rats, respectively. We scrutinized the cellular correlates and mechanisms underlying the emergence of these two forms of HFOs by combining multisite, single-cell and paired-cell recordings in slices prepared from a rat reporter line that facilitates identification of GABAergic cells. We found a major effect of extracellular Ca(2+) in modulating intrinsic excitability and disynaptic inhibition, two critical factors shaping network dynamics. Moreover, locally modulating the extracellular Ca(2+) concentration in an in vivo environment had a similar effect on disynaptic inhibition, pyramidal cell excitability, and ripple dynamics. Therefore, the HFO frequency band reflects a range of firing dynamics of hippocampal networks.

SU-8 based microprobes for simultaneous neural depth recording and drug delivery in the brain.

While novel influential concepts in neuroscience bring the focus to local activities generated within a few tens of cubic micrometers in the brain, we are still devoid of appropriate tools to record and manipulate pharmacologically neuronal activity at this fine scale. Here we designed, fabricated and encapsulated microprobes for simultaneous depth recording and drug delivery using exclusively the polymer SU-8 as structural material. A tetrode- and linear-like electrode patterning was combined for the first time with single and double fluidic microchannels for independent drug delivery. The device was tested experimentally using the in vivo anesthetized rat preparation. Both probe types successfully recorded detailed spatiotemporal features of local field potentials and single-cell activity at a resolution never attained before with integrated fluidic probes. Drug delivery was achieved with high spatial and temporal precision in a range from tens of nanoliters to a few microliters, as confirmed histologically. These technological advancements will foster a wide range of neural applications aimed at simultaneous monitoring of brain activity and delivery at a very precise micrometer scale.

Basic properties of somatosensory-evoked responses in the dorsal hippocampus of the rat.

The hippocampus is a pivotal structure for episodic memory function. This ability relies on the possibility of integrating different features of sensory stimuli with the spatio-temporal context in which they occur. While recent studies now suggest that somatosensory information is already processed by the hippocampus, the basic mechanisms still remain unexplored. Here, we used electrical stimulation of the paws, the whisker pad or the medial lemniscus to probe the somatosensory pathway to the hippocampus in the anaesthetized rat, and multisite electrodes, in combination with tetrode and intracellular recordings, to look at the properties of somatosensory hippocampal responses. We found that peripheral and lemniscal stimulation elicited small local field potential responses in the dorsal hippocampus about 35-40 ms post-stimulus. Current source density analysis established the local nature of these responses, revealing associated synaptic sinks that were consistently confined to the molecular layer (ML) of the dentate gyrus (DG), with less regular activation of the CA1 stratum lacunosum moleculare (SLM). A delayed (40-45 ms), potentially active, current source that outlasted the SLM sink was present in about 50% cases around the CA1 pyramidal cell layer. Somatosensory stimulation resulted in multi-unit firing increases in the majority of DG responses (79%), whereas multi-unit firing suppression was observed in the majority of CA1 responses (62%). Tetrode and intracellular recordings of individual cells confirmed different firing modulation in the DG and the CA1 region, and verified the active nature of both the early ML sink and delayed somatic CA1 source. Hippocampal responses to somatosensory stimuli were dependent on fluctuations in the strength and composition of synaptic inputs due to changes of the ongoing local (hippocampal) and distant (cortical) state. We conclude that somatosensory signals reach the hippocampus mainly from layer II entorhinal cortex to directly discharge DG granule cells, while a different predominantly inhibitory process takes place in CA1, further controlling the hippocampal output. Therefore, our data reveal a distinct organization of somatosensory-related extra-hippocampal inputs converging onto DG and CA1.

SU-8 based microprobes with integrated planar electrodes for enhanced neural depth recording.

Here, we describe new fabrication methods aimed to integrate planar tetrode-like electrodes into a polymer SU-8 based microprobe for neuronal recording applications. New concepts on the fabrication sequences are introduced in order to eliminate the typical electrode-tissue gap associated to the passivation layer. Optimization of the photolithography technique and high step coverage of the sputtering process have been critical steps in this new fabrication process. Impedance characterization confirmed the viability of the electrodes for reliable neuronal recordings with values comparable to commercial probes. Furthermore, a homogeneous sensing behavior was obtained in all the electrodes of each probe. Finally, in vivo action potential and local field potential recordings were successfully obtained from the rat dorsal hippocampus. Peak-to-peak amplitude of action potentials ranged from noise level to up to 400-500 µV. Moreover, action potentials of different amplitudes and shapes were recorded from all the four recording sites, suggesting improved capability of the tetrode to distinguish from different neuronal sources.

Statistical analysis of genomic protein family and domain controlled annotations for functional investigation of classified gene lists.

BACKGROUND: The increasing protein family and domain based annotations constitute important information to understand protein functions and gain insight into relations among their codifying genes. To allow analyzing of gene proteomic annotations, we implemented novel modules within GFINDer, a Web system we previously developed that dynamically aggregates functional and phenotypic annotations of user-uploaded gene lists and allows performing their statistical analysis and mining. RESULTS: Exploiting protein information in Pfam and InterPro databanks, we developed and added in GFINDer original modules specifically devoted to the exploration and analysis of functional signatures of gene protein products. They allow annotating numerous user-classified nucleotide sequence identifiers with controlled information on related protein families, domains and functional sites, classifying them according to such protein annotation categories, and statistically analyzing the obtained classifications. In particular, when uploaded nucleotide sequence identifiers are subdivided in classes, the Statistics Protein Families&Domains module allows estimating relevance of Pfam or InterPro controlled annotations for the uploaded genes by highlighting protein signatures significantly more represented within user-defined classes of genes. In addition, the Logistic Regression module allows identifying protein functional signatures that better explain the considered gene classification. CONCLUSION: Novel GFINDer modules provide genomic protein family and domain analyses supporting better functional interpretation of gene classes, for instance defined through statistical and clustering analyses of gene expression results from microarray experiments. They can hence help understanding fundamental biological processes and complex cellular mechanisms influenced by protein domain composition, and contribute to unveil new biomedical knowledge about the codifying genes.

Genomic functional investigation through statistical analysis of protein families and domains.

Protein families and domains represent a very relevant resource useful to understand protein functions and interactions among their codifying genes. To perform evaluations of gene annotations sparsely available in numerous different databanks accessible via Internet, we previously developed GFINDer, a Web server that performs statistical analysis of functional and phenotypic annotations of gene lists. To exploit protein information present in Pfam and InterPro databanks, in GFINDer we integrated two new modules, that allow to annotate and statistically analyze user-classified nucleotide sequence with controlled information on related protein families, domains and functional sites.