Recent advances in three-dimensional (3D) biological brain models in vitro and ex vivo are creating new opportunities to understand the complexity of neural networks but pose the technological challenge of obtaining high-throughput recordings of electrical activity from multiple sites in 3D at high spatiotemporal resolution. This cannot be achieved using planar multi-electrode arrays (MEAs), which contact just one side of the neural structure. Moreover, the specimen adhesion to planar MEAs limits fluid perfusion along with tissue viability and drug application. Here, the efficiency of the tissue-sensor interface provided by advanced 3D high-density (HD)-MEA technology was evaluated in acute brain slices, spheroids, and organoids obtained from different brain regions. The 3D HD-MEA microneedles reached the inner layers of samples without damaging network integrity and the microchannel network between microneedles improved tissue vitality and chemical compound diffusion. In acute cortico-hippocampal and cerebellar slices, signal recording and stimulation efficiency proved higher with the 3D HD-MEA than with a planar MEA improving the characterization of network activity and functional connectivity. The 3D HD-MEA also resolved the challenge of recording from brain spheroids as well as cortical and spinal organoids. Our results show that 3D HD-MEA technology represents a valuable tool to address the complex spatiotemporal organization of activity in brain microcircuits, making it possible to investigate 3D biological models.

We invited contributions reporting new evidence of synaptic mechanisms and their network-level impacts for this Special Issue [...]

In the last decades, planar multi-electrode arrays (MEAs) have been widely used to record activity from in vitro neuronal cell cultures and tissue slices. Though successful, this technique bears some limitations, particularly relevant when applied to three-dimensional (3D) tissue, such as brain slices, spheroids or organoids. For example, planar MEAs signals are informative on just one side of a 3D-organized structure. This limits the interpretation of the results in terms of network functions in a complex structured and hyperconnected brain tissue. Moreover, the side in contact with the MEAs often shows lower oxygenation rates and related vitality issues. To overcome these problems, we empowered a CMOS high-density multi-electrode array (HD-MEA) with thousands of microneedles (μneedles) of 65-90 μm height, able to penetrate and record in-tissue signals, providing for the first time a 3D HD-MEA chip. We propose a CMOS-compatible fabrication process to produce arrays of μneedles of different widths mounted on large pedestals to create microchannels underneath the tissue. By using cerebellar and cortico-hippocampal slices as a model, we show that the μneedles efficiently penetrate the 3D tissue while the microchannels allow the flowing of maintenance solutions to increase tissue vitality in the recording sites. These improvements are reflected by the increase in electrodes sensing capabilities, the number of sampled neuronal units (compared to matched planar technology), and the efficiency of compound effects. Importantly, each electrode can also be used to stimulate the tissue with optimal efficiency due to the 3D structure. Furthermore, we demonstrate how the 3D HD-MEA can efficiently penetrate and get outstanding signals from in vitro 3D cellular models as brain spheroids. In conclusion, we describe a new recording device characterized by the highest spatio-temporal resolution reported for a 3D MEA and significant improvements in the quality of recordings, with a high signal-to-noise ratio and improved tissue vitality. The applications of this game-changing technique are countless, opening unprecedented possibilities in the neuroscience field and beyond.### Competing Interest StatementMariateresa Tedesco, Giacomo Sciacca, and Chiara Battaglia are employees of 3Brain AG. Mauro Gandolfo, Kilian Imfeld, and Alessandro Maccione are shareholders of 3Brain AG. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Golgi cells, together with granule cells and mossy fibers, form a neuronal microcircuit regulating information transfer at the cerebellum input stage. Despite theoretical predictions, little was known about long-term synaptic plasticity at Golgi cell synapses. Here, we have used whole-cell patch-clamp recordings and calcium imaging to investigate long-term synaptic plasticity at excitatory synapses impinging on Golgi cells. In acute mouse cerebellar slices, mossy fiber theta-burst stimulation (TBS) could induce either long-term potentiation (LTP) or long-term depression (LTD) at mossy fiber-Golgi cell and granule cell-Golgi cell synapses. This synaptic plasticity showed a peculiar voltage dependence, with LTD or LTP being favored when TBS induction occurred at depolarized or hyperpolarized potentials, respectively. LTP required, in addition to NMDA channels, activation of T-type Ca2+ channels, while LTD required uniquely activation of L-type Ca2+ channels. Notably, the voltage dependence of plasticity at the mossy fiber-Golgi cell synapses was inverted with respect to pure NMDA receptor-dependent plasticity at the neighboring mossy fiber-granule cell synapse, implying that the mossy fiber presynaptic terminal can activate different induction mechanisms depending on the target cell. In aggregate, this result shows that Golgi cells show cell-specific forms of long-term plasticity at their excitatory synapses, that could play a crucial role in sculpting the response patterns of the cerebellar granular layer.SIGNIFICANCE STATEMENT This article shows for the first time a novel form of Ca2+ channel-dependent synaptic plasticity at the excitatory synapses impinging on cerebellar Golgi cells. This plasticity is bidirectional and inverted with respect to NMDA receptor-dependent paradigms, with long-term depression (LTD) and long-term potentiation (LTP) being favored at depolarized and hyperpolarized potentials, respectively. Furthermore, LTP and LTD induction requires differential involvement of T-type and L-type voltage-gated Ca2+ channels rather than the NMDA receptors alone. These results, along with recent computational predictions, support the idea that Golgi cell plasticity could play a crucial role in controlling information flow through the granular layer along with cerebellar learning and memory.

The deep cerebellar nuclei (DCN) have been suggested to play a critical role in sensorimotor learning and some forms of long-term synaptic plasticity observed in vitro have been proposed as a possible substrate. However, till now it was not clear whether and how DCN neuron responses manifest long-lasting changes in vivo. Here, we have characterized DCN unit responses to tactile stimulation of the facial area in anesthetized mice and evaluated the changes induced by theta-sensory stimulation (TSS), a 4 Hz stimulation pattern that is known to induce plasticity in the cerebellar cortex in vivo. DCN units responded to tactile stimulation generating bursts and pauses, which reflected combinations of excitatory inputs most likely relayed by mossy fiber collaterals, inhibitory inputs relayed by Purkinje cells, and intrinsic rebound firing. Interestingly, initial bursts and pauses were often followed by stimulus-induced oscillations in the peri-stimulus time histograms (PSTH). TSS induced long-lasting changes in DCN unit responses. Spike-related potentiation and suppression (SR-P and SR-S), either in units initiating the response with bursts or pauses, were correlated with stimulus-induced oscillations. Fitting with resonant functions suggested the existence of peaks in the theta-band (burst SR-P at 9 Hz, pause SR-S at 5 Hz). Optogenetic stimulation of the cerebellar cortex altered stimulus-induced oscillations suggesting that Purkinje cells play a critical role in the circuits controlling DCN oscillations and plasticity. This observation complements those reported before on the granular and molecular layers supporting the generation of multiple distributed plasticities in the cerebellum following naturally patterned sensory entrainment. The unique dependency of DCN plasticity on circuit oscillations discloses a potential relationship between cerebellar learning and activity patterns generated in the cerebellar network.

The induction of long-term potentiation and depression (LTP and LTD) is thought to trigger gene expression and protein synthesis, leading to consolidation of synaptic and neuronal changes. However, while LTP and LTD have been proposed to play important roles for sensori-motor learning in the cerebellum granular layer, their association with these mechanisms remained unclear. Here, we have investigated phosphorylation of the cAMP-responsive element binding protein (CREB) and activation of the immediate early gene c-Fos pathway following the induction of synaptic plasticity by theta-burst stimulation (TBS) in acute cerebellar slices. LTP and LTD were localized using voltage-sensitive dye imaging (VSDi). At two time points following TBS (15 min and 120 min), corresponding to the early and late phases of plasticity, slices were fixed and processed to evaluate CREB phosphorylation (P-CREB) and c-FOS protein levels, as well as Creb and c-Fos mRNA expression. High levels of P-CREB and Creb/c-Fos were detected before those of c-FOS, as expected if CREB phosphorylation triggered gene expression followed by protein synthesis. No differences between control slices and slices stimulated with TBS were observed in the presence of an N-methyl-D-aspartate receptor (NMDAR) antagonist. Interestingly, activation of the CREB/c-Fos system showed a relevant degree of colocalization with long-term synaptic plasticity. These results show that NMDAR-dependent plasticity at the cerebellum input stage bears about transcriptional and post-transcriptional processes potentially contributing to cerebellar learning and memory consolidation.

The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

The consolidation of changes following activity-dependent neural plasticity are believed to involve specific patterns of gene expression. In the hippocampus, immediate early genes are thought to contribute to long-term synaptic plasticity (LTP and LTD); this phenomenon may occur also in the cerebellum, in which the transcription factors c-Fos and P-CREB have been identified. The cerebellum granular layer (GL) can manifest both LTP and LTD following a Theta Burst Stimulus (TBS) delivered to the mossy fibers. We have employed VSD imaging in rat cerebellar slices (P18-24) in order to map the spatial distribution of LTP and LTD in the cerebellum GL. Fluorescence changes were correlated to LTP or LTD in two different post-TBS time ranges (15 and 120 min). Slices were then fixed and processed for immunohistochemistry in order to identify levels of c-Fos and P-CREB expression. The induction of long-term plasticity increased the average level of P-CREB both at 15 min (+39±4.9, p<0.01%) and 120 min (+24±7.2, p<0.05%) after TBS. The level of c-Fos was unaltered at 15 min, while it significantly increased at 120 min (+37±8.9, p<0.05%). By spatially correlating longterm synaptic plasticity with the corresponding variation of P-CREB and c-Fos, we observed that regions showing LTP well correlated (p<0.05) with positive variations of P-CREB and c-Fos. Conversely, areas showing LTD correlated exclusively (p<0.05) with negative variations of P-CREB. Slices were also evaluated by in situ hybridization and a similar analysis was performed. The levels of fos and CREB mRNA expression and their spatial correlation with the sign of long-term synaptic plasticity corresponded with the immunohistochemical results. As a further test, VSD recordings showed that the induction of granular layer LTP and LTD could be prevented by applying 50 mM D-APV, a selective NMDA receptor blocker. Moreover, in situ hybridization and immunohistochemistry analysis evidenced that in these conditions both mRNA and protein expression levels of c-fos and CREB were unchanged, confirming the involvement of these two transcription factors in cerebellar granular layer plasticity.

Several experiments suggest an important role for store-released Ca 2+ in hair cell organs: drugs targeting IP 3 and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyR α in both ampulla and canal arm and RyR β in the ampulla only.

The cerebellum is one of the brain areas involved in learning and memory formation. Long-term synaptic plasticity is thought to play a pivotal role in supporting these functions. Moreover Immediate Early Genes (IEGs) expression and de novo protein synthesis and/or modification have been strictly associated with maintenance of Long-Term Potentiation (LTP) as well as memory consolidation and storage. Two highly conserved signalling cascades, PKA and MAPK, seem to be involved in early- to late-LTP conversion; both pathway can activate CREB transcription factor through phosphorylation and P-CREB has been suggested to initiate the protein synthesis leading to late-LTP induction. The transcription factor c-fos is known to be rapidly and transiently induced in the Nervous System by a variety of stimuli and is thought to be directly involved in processes of neuronal plasticity including LTP. We used rat parasagittal cerebellar slices as a model system in which specific patterns of stimulation delivered to the mossy fibers can induce both Long-Term Potentiation and Long-Term Depression (LTD), depending on local inhibition and other regulating factors. Using Voltage Sensitive Dye (VSD) imaging we obtained high-resolution maps of the spatial distribution of LTP/LTD induced from a Teta Burst Stimulus (TBS) application. Control and stimulated slices were fixed at different times from the TBS application and processed for in situ hybridization or immunohystochemistry in order to detect IEGs mRNA expression patterns and protein expression/modifications. The expression pattern of c-fos and CREB mRNAs and their protein distribution and/or phosphorylation were then correlated with LTP/LTD maps generated by VSD imaging. Preliminary data indicate a significant increase of P-CREB in the granular layer suggesting that CREB phosphorylation is induced as early as 15 minutes post TBS application. In situ hybridization experiments indicate a good correlation between c-fos and CREB mRNAs up-regulation and LTP distribution at 120 minutes post TBS. At the protein level, the comparison of immunofluorescence signals and VSD immaging data indicate a clear correlation between c-Fos and P-CREB distribution and synaptic plasticity patterns. We are planning further experiments to confirm these data and to test our experimental system in the presence of drugs that could interfere with the transcription, translation or post-translational protein regulation.

We characterize the expression pattern of aquaporin-6 in the mouse inner ear by RT-PCR and immunohistochemistry. Our data show that in the inner ear aquaporin-6 is expressed, in both vestibular and acoustic sensory epithelia, by the supporting cells directly contacting hair cells. In particular, in the Organ of Corti, expression was strongest in Deiters' cells, which provide both a mechanical link between outer hair cells (OHCs) and the Organ of Corti, and an entry point for ion recycle pathways. Since aquaporin-6 is permeable to both water and anions, these results suggest its possible involvement in regulating OHC motility, directly through modulation of water and chloride flow or by changing mechanical compliance in Deiters' cells. In further support of this role, treating mice with salicylates, which impair OHC electromotility, dramatically reduced aquaporin-6 expression in the inner ear epithelia but not in control tissues, suggesting a role for this protein in modulating OHCs' responses.

BACKGROUND:Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. The site of action of these drugs however has not been elucidated yet. Recent works on amphibians showed that histamine H3 receptor antagonists, e.g. betahistine, inhibit the afferent discharge recorded from the vestibular nerve. To assess the expression of H3 histamine receptors in vestibular neurons, we performed mRNA RT-PCR and immunofluorescence experiments in mouse Scarpa's ganglia.RESULTS:RT-PCR analysis showed the presence of H3 receptor mRNA in mouse ganglia tissue. H3 protein expression was found in vestibular neurons characterized by large and roundish soma, which labeled for calretinin and calbindin.CONCLUSION:The present results are consistent with calyx and dimorphic, but not bouton, afferent vestibular neurons expressing H3 receptors. This study provides a molecular substrate for the effects of histamine-related antivertigo drugs acting on (or binding to) H3 receptors, and suggest a potential target for the treatment of vestibular disorders of peripheral origin.

BACKGROUND:Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.RESULTS:RT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding. Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein. The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells.CONCLUSION:The results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. Their site and mechanism of action, however, are still poorly understood. To increase our knowledge of the histaminergic system in the vestibular organs, we have investigated the expression of H1 and H3 histamine receptors in the frog and mouse semicircular canal sensory epithelia. Analysis was performed by mRNA reverse transcriptase-PCR, immunoblotting and immunocytochemistry experiments. Our data show that both frog and mouse vestibular epithelia express H1 receptors. Conversely no clear evidence for H3 receptors expression was found.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (P f ) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg 2+ , Cu 2+ , and Zn 2+ caused a significant decrease in the BBMV P f , whereas Ni 2+ and Li + had no effect. AQP8-transfected cells showed a reduction in P f in the presence of Hg 2+ and Cu 2+ , whereas AQP7-transfected cells were insensitive to all tested metals. The P f of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the P f of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg 2+ and Cu 2+ .

Background. Tissue transglutaminase, the coeliac autoantigen, was shown to localise in the enterocytes of coeliac patients and controls. It was speculated that surface tissue transglutaminase has a role in the pathogenesis of coeliac disease.Aims. To study localisation of tissue transglutaminase in different stages of coeliac disease and other enteropathies with and without villous flattening.Methods. Imunofluorescent and immunoblotting assays were used. Duodenal cryostat sections from 23 coeliac patients (10 untreated, 8 treated, 5 potential) and 18 controls (2 autoimmune enteropathy and 16 normal duodenal mucosa) were incubated with an anti-tissue transglutaminase monoclonal antibody. Slides were blindly examined.Results. The immunofluorescent assay showed that monoclonal antibody localised in the subepithelial layer, in the lamina propria, and in the pericryptal connective tissue of all samples. It also bound to surface enterocytes in 8/10 untreated, 1/8 treated, and 3/5 potential coeliac patients. None of the controls showed an epithelial distribution of tissue transglutaminase. Immunoblotting experiments performed in enterocytes freshly isolated from duodenal biopsy confirmed these findings.Conclusion. Epithelial distribution of tissue transglutaminase is specific for coeliac disease rather than due to a non-specific mucosal inflammation. Analysis of different stages of coeliac disease suggests that the epithelial distribution of tissue transglutaminase is gluten dependent. (c) 2006 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.