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Interferon-stimulated Gene 15 (ISG15) and ISG15-linked Proteins Can Associate with Members of the Selective Autophagic Process, Histone Deacetylase 6 (HDAC6) and SQSTM1/p62*

Background: ISG15 is a Ub-like protein that conjugates cellular and pathogenic proteins during the innate immune response. Results: ISG15 associates with the selective autophagic factors HDAC6 and p62, leading to degradation of ISG15 conjugates. Conclusion: The IFN response leads to ISG15 expression allowing for its association with HDAC and p62. This may mark proteins for autophagy. Significance: This finding provides evidence of an interferon-stimulated pathway linked to autophagy. The ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) and its specific E1, E2, and E3 enzymes are transcriptionally induced by type I IFNs. ISG15 conjugates newly synthesized proteins. ISG15 linkage to proteins appears to be an important downstream IFN signaling event that discriminates cellular and pathogenic proteins synthesized during IFN stimulation from existing proteins. This eliminates potentially pathogenic proteins as the cell attempts to return to normal homeostasis after IFN “stressed” conditions. However, the molecular events that occur in this process are not well known. Here, we show that the C-terminal LRLRGG of ISG15 interacts with the binder of ubiquitin zinc finger (BUZ) domain of histone deacetylase 6 (HDAC6). Because HDAC6 is involved in the autophagic clearance of ubiquitinated aggregates during which SQSTM1/p62 plays a major role as a cargo adapter, we also were able to confirm that p62 binds to ISG15 protein and its conjugated proteins upon forced expression. Both HDAC6 and p62 co-localized with ISG15 in an insoluble fraction of the cytosol, and this co-localization was magnified by the proteasome inhibitor MG132. In addition, ISG15 was degraded via the lysosome. Overexpression of ISG15, which leads to an increased conjugation level of the cellular proteome, enhanced autophagic degradation independently of IFN signaling transduction. These results thus indicate that ISG15 conjugation marks proteins for interaction with HDAC6 and p62 upon forced stressful conditions likely as a step toward autophagic clearance.

understood. The 26 S proteasome, which is responsible for degrading ubiquitinated proteins as part of the canonical ubiquitin-proteasome system (UPS), is not thought to play a role in degradation of ISG15 conjugates (5,6).
Although in general the UPS is involved primarily in the degradation of single molecules, the lysosome in the process of (macro-) autophagy is more involved in eliminating large multiple molecules, organelles, and microbes/viruses. Type I interferons have also been reported to induce autophagy, which plays a role in cellular homeostasis through sequestration of intracellular materials including pathogens within double membrane vesicles (autophagosomes) that fuse with lysosomes, leading to degradation of delivered cellular contents (7). Although starvation-induced, non-selective autophagy recycles cytosolic contents and organelles during limited nutrient supply, nutrient-independent, basal, or selective autophagy eliminates misfolded proteins and damaged organelles before the accumulation of excessive amounts of cytosolic aggregates or inclusion bodies at the microtubule-organizing center (8 -10). It has been reported that aggregate-prone proteins become marked by the lysine 63 (Lys 63 )-linked polyubiquitin chain that functionally distinguishes them from UPS-targeted lysine 48 (Lys 48 )-linked polyubiquitin chains (11,12). In this context, the cargo adaptor protein SQSTM1/p62 plays an important role by preferentially binding Lys 63 -linked polyubiquitinated proteins via a ubiquitin-associated domain at its C terminus. To facilitate ubiquitin (Ub)-protein aggregates and autophagic degradation, p62 undergoes self-and/or heterooligomerization at its N-terminal PB1 domain and recruits the LC3 membrane protein that is anchored in phagophore membranes for autophagosome maturation (13)(14)(15). During selective autophagy, the cytosolic deacetylase HDAC6 is involved in clearance of ubiquitin-prone aggregates (10,16). Its C-terminal binder of ubiquitin zinc finger (BUZ) domain can bind free mono-and poly-Ub chains (17,18). Both the catalytic activity and the BUZ domain are required for HDAC6 to facilitate clearance of ubiquitin-prone aggregates (also referred to as "aggrephagy"), and HDAC6 promotes autophagosome-lysosome fusion through the cortactin-dependent actin remodeling machinery in mouse embryonic fibroblast cells (19).
Here, we report that forced expression of HDAC6 leads to interaction with p62 and ISG15 that structurally resembles Lys 63 -di-Ub and its ISGylated substrates, rendering them targets for lysosomal degradation. ISG15 localized to cytosolic inclusion bodies with HDAC6 and p62. Pharmacological induction of aggrephagy became more prominent if ISG15 was overexpressed. Therefore, our results suggest that ISG15 augments p62-mediated aggresome formation and their autophagic degradation under conditions of cellular stress such as forced expression of genes, implying an important role during intrinsic cellular defense.

Materials
Reagents-Human IFN␤ 1a (11410) was obtained from PBL Assay Science. Proteasome inhibitor MG132 (40 mM stock in DMSO) was from EMD Millipore. Lysosomal protease inhibi-tors pepstatin A (20 mM in DMSO) and E64d (10 mg/ml in DMSO) were from Enzo Life Science. Tubacin was kindly obtained from Dr. Stuart Schreiber (Broad Institute of Massachusetts Institute of Technology/Harvard, Cambridge, MA) (20). Doxycycline hyclate was purchased from Sigma. Protease inhibitor mixture Complete EDTA-free and phosphatase inhibitor mixture PhosSTOP were from Roche Applied Science. Recombinant GST-tagged HDAC6 protein was from EMD Millipore. GST-tagged p62 was from Enzo Life Science. Human ISG15 protein was from Boston Biochem. Anti-FLAG M1-agarose affinity gel and monoclonal anti-FLAG M1 antibody were from Sigma-Aldrich. Immobilized GST-agarose and GST protein were from Thermo Fisher Scientific.

DNA Constructs
For mutant constructs, HDAC6 point mutation at histidine 611 to alanine (H611A) and deletions from residues 841 to 1195 (⌬Cter), from 2 to 837 (⌬CD1/2), from 841 to 1053 (⌬SE14), and from 1133 to 1195 (⌬BUZ) were generated from HDAC6 Flag plasmid DNA by PCR (QuikChange site-directed mutagenesis kit, Agilent Technologies). For lentivirus packaging, some HDAC6 constructs were also cloned in pGIPZ at blunt-ended NotI and XbaI sites by inserting the SpeI-XbaI fragments that contain a CMV intermediate-early promoter and an HDAC6 coding sequence. ISG15 truncated mutants in the C-terminal end peptides LRLRGG and aspartic acid (D) insert (LRLRGGD) were also generated from a pcDNA-hISG15 plasmid by PCR. The sequences of all mutant constructs were verified by DNA sequencing analyses. For the ISG15-cyan fluorescent protein (CFP) construct, three repeated FLAG sequences were added in ISG15 by PCR using the primers 5Јcaccctcgagctccatatggactacaaagaccatgacggtgatta-3Ј and 5Ј-ttaggatcccgggcccgcccacccctcaggcgcaga-3Ј and pcDNA-hISG15 as template. These were cloned in pENTR/D-TOPO (Invitrogen). The FLAG-ISG15 fragment was inserted in pmTurquoise2-Golgi (Addgene, 36205) at XhoI/BamHI sites. FLAG-tagged mTurquoise2 (referred to CFP in this study) was engineered previously. 3 Gene transfer was performed either by Lipofectoamine 2000 (for 293), Lipofectoamine 3000 (for U251), or lentiviral infection. For transduction of genes via lentiviral infection, vectors were packaged in lentiviral vector systems using 293T (ATCC) or 293FT cells (Invitrogen) by co-transfection with packaging vectors psPAX2 and pMD2.G (a gift from Dr. Didier Trono, Switzerland) followed by lentivirus infection. U251 cells expressing FLAG-HDAC6 wild type (WT) and derivative mutants were stably selected by G418 treatment after infection.

Protein Soluble and Insoluble Fractions
Cells were lysed in soluble lysis buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, 0.5% Triton X-100, 40 M MG132, PhosSTOP, and protease inhibitor mixture) and incubated on ice for 5 min. After centrifugation at 13,000 rpm, the supernatant was collected in the tube as the soluble fraction, whereas the pellet was dissolved in insoluble lysis buffer (1% SDS in soluble lysis buffer) followed by sonication and centrifugation to collect the supernatant as the insoluble fraction.

Protein Interaction Assays
For the in vitro GST pulldown assay of p62, each 2 g of recombinant GST-p62 or GST protein was incubated with immobilized glutathione-agarose in binding buffer (20 mM Tris-Cl (pH 7.4), 50 mM NaCl, 0.1% Nonidet P-40, 25 g/ml BSA, 1 mM ATP, 20 M MG132, and protease inhibitor mixture) followed by three washes with binding buffer. The recombinant mature form of human ISG15 protein (2 g) was incubated with GST protein-bound agarose overnight at 4°C before analysis by immunoblotting. The GST-HDAC6 pulldown assay was performed using the cell lysate of 293T cells that were transfected with mutant ISG15 constructs. For the co-immunoprecipitation assay, cells were lysed in a buffer containing 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM EGTA, 40 M MG132, PhosSTOP, and protease inhibitor mixture. The lysates were incubated with anti-FLAG M2 affinity gel overnight at 4°C. Immunocomplexes were collected by centrifugation and analyzed by immunoblotting.

Immunofluorescence Microscopy Analysis
Cells were cultured on German glass round coverslip (number 1 thickness) and then washed with 37°C PBS once before fixation with 4% paraformaldehyde in PBS. For prepermeabilized cell preparation, washed cells were incubated in extraction buffer (100 mM PIPES (pH 6.9), 0.5 mM MgCl 2 , 0.1 mM EDTA, and 0.5% Triton X-100) for ϳ20 s before 4% paraformaldehyde fixation. The samples were quenched with 50 mM NH 4 Cl in PBS and permeabilized with 50 g/ml digitonin (EMD Millipore) for the non-prepermeabilized condition. The samples were washed with 0.1% gelatin in PBS and incubated with diluted antibodies (1:200, ␣-tubulin and FLAG M; 1:50, p62 and ISG15) followed by Alexa Fluor antibodies (1:200; Invitrogen). Nuclei were counterstained with DAPI during the wash step, and then coverslips were mounted with VectaShield. Fluorescence images were acquired with a Zeiss LSM510 META or LSM710 confocal microscopy system and processed using NIH ImageJ (1.48 or earlier version). Figure panels were assembled using Adobe Illustrator CS5.

HDAC6 Associates with IFN-induced ISG15-conjugated
Proteins-We first attempted to analyze whether an interaction between ISG15-conjugated substrates and HDAC6 occurred upon IFN treatment. We attempted to utilize two commercially available antibodies raised against endogenous HDAC6 to detect co-immunoprecipitates of HDAC6 with ISG15 conjugates in U251 exposed to type I IFN. However, we were unable to visualize co-immunoprecipitation of endogenous HDAC6 with ISGylated substrates (data not shown). Although there could be multiple reasons for this, we reasoned that perhaps we needed to overexpress HDAC6 and/or the HDAC6 antibody was not sufficiently avid to allow co-immunoprecipitations.
We thus performed a second series of experiments whereby the lysates of human U251 glioma cells forced to express FLAGtagged HDAC6 by lentivirus transduction were immunoprecipitated with an antibody against FLAG-peptides and the coimmunoprecipitates of FLAG-HDAC6 were analyzed by Western blotting using antibodies against ISG15 or ubiquitin. Fig. 1a shows that IFN␤ treatment led to ISG15 conjugation. FLAG-HDAC6 co-immunoprecipitated with ISGylated substrates, and this was even more prominent when cells were treated with MG132, a protease inhibitor that induces forma-tion of intracellular aggresomes. In contrast, in comparison with ISGylated substrates, there was a specific effect of MG132 on FLAG-HDAC6 association with ubiquitinated substrates that was independent of IFN treatment (17,18). These data thus suggested that there was a previously undescribed but visible association of forcibly expressed FLAG-HDAC6 with IFN␤induced ISGylated substrates even in the absence of MG132 (i.e. when the UPS is functional).
Next, we visualized the subcellular localization of FLAG-HDAC6 and ISG15 in IFN␤-treated cells utilizing immunofluorescence staining with antibodies against FLAG and ISG15 (Fig. 1b). ISG15 displayed small punctate stainings in the cytoplasm with a concentration in the perinuclear region where FLAG-HDAC6 vacuoles also appeared to partially localize, although co-localization was minimal if any. To examine whether this lack of localization reflected insoluble aggregates, live cells were permeabilized with a pulse of Triton X-100 to remove soluble proteins from cytosols prior to fixation (Fig. 1, c and d). In permeabilized cells in the absence of proteasomal inhibition, ISG15 punctate stainings were visualized in the same perinuclear region as that of small HDAC6 subfractions (Fig. 1c). However, in the presence of MG132 proteasome inhibition, there was visually intensified HDAC6 immunofluorescence with a perinuclear aggregation pattern that now also colocalized with ISG15 speckles (Fig. 1d). Taken together, these results suggested that the interaction between HDAC6 and ISG15 was localized to detergent-resistant aggregates in the perinuclear region and that proteasome inhibition led to a prominent co-localization of ISG15 and HDAC6 in the same aggregates.
HDAC6 Binds ISG15 at the C-terminal LRLRGG Motif via a BUZ Domain-To find out which motif of HDAC6 interacts with ISG15, we engineered U251 cells that stably overexpress FLAG-tagged full-length HDAC6 WT or its C-terminal deletion mutant (⌬Cter; ⌬841-1195) or its catalytic subsite mutant (H611A) (Fig. 2a). Subsequently, we performed co-immunoprecipitation assays with an antibody against FLAG peptides using cell lysates that had been treated with IFN␤. Fig. 2b shows that ISG15 was bound to FLAG-HDAC6 WT and FLAG-HDAC6H611A but not to FLAG-HDAC6⌬Cter. To further map the ISG15 binding site in residues 841-1195 of the HDAC6 C-terminal region, we transiently transfected FLAGtagged full-length HDAC6 and truncation mutant constructs of HDAC6 that lacked the BUZ domain (⌬BUZ), the Ser-Glucontaining tetradecapeptide repeat (SE14) domain (⌬SE14), or both catalytic domains (⌬CD1/2) (see Fig. 2a) together with the HA-tagged mature form of ISG15 in 293 cells. In addition to FLAG-HDAC6 WT, Fig. 2c shows that ISG15 co-immunoprecipitated with ⌬CD1/2 and ⌬SE14 but not with the ⌬BUZ truncation mutant of HDAC6. Therefore, these findings indicated that residues 1133-1195, corresponding to a BUZ domain, were responsible for ISG15 binding.
Based on this, we then sought to find the motif in ISG15 responsible for binding to HDAC6. It has been reported that the BUZ domain of HDAC6 interacts with a terminal conjugation motif of Ub composed of LRLRGG. This motif appears in Ub aggregates that contain free mono-or polyubiquitin chains (22). The mature form of ISG15 also has a C-terminal LRLRGG motif, and thus we asked whether it also bound to HDAC6 BUZ. We utilized a full-length mature ISG15 (ISG15 WT) and four mutants of ISG15 (ISG15-LRLRG, ISG15-LRLR, ISG15-LRLRGGD, and ISG15-LRGG) with deletions or additions at the C-terminal residues (Fig. 2d). In vitro protein affinity assays revealed that the full-length, wild-type LRLRGG motif was the most efficient at interacting with recombinant GST-tagged HDAC6, whereas all the other mutant forms of ISG15 failed to bind to GST-HDAC6 (Fig. 2e). There was some relatively weak interaction of the LRLRGGD mutant with HDAC6. Overall, these findings thus confirmed that the ISG15 LRLRGG peptide motif is indispensable for binding to the BUZ domain of HDAC6.
ISG15 Associates with p62 Likely to Facilitate Autophagosome/Lysosome Clearance-The Ub binding property of HDAC6 is associated with formation and clearance of organized aggregates (also called aggresomes) as part of the autophagy-lysosome degradation pathway (12,17,23  this process, the SQSTM1/p62 cargo protein forms aggregates of ubiquitinated proteins and recruits LC3 anchored in the phagophore membrane (24). p62 preferentially binds with higher affinity to Lys 63 -linked di-Ub when compared with binding to monoubiquitin and Lys 48 -linked di-Ub (25). Our in silico analysis using the flexible structure alignment (available on the Protein Data Bank site) predicted that the structure of ISG15 (Protein Data Bank code 1z2m) resembles that of Lys 63 -linked di-Ub (Protein Data Bank code 2w9n) rather than that of Lys 48linked di-Ub (Protein Data Bank code 3m3j) (26 -29). We thus sought to determine whether ISG15 also bound to p62. In fact, a pulldown assay using recombinant GST-tagged p62 did lead to direct binding of GST-p62 to ISG15 (Fig. 3a). This indicated that ISG15, like Lys 63 -linked di-Ub, also directly bound to p62. To test whether p62 was involved in the formation and clearance of aggregates of ISGylated proteins, we engineered U251 cells that expressed a Dox-inducible transcriptionally regulated ISG15 as well as UbE1L, UbcH8, and Herc5, the enzymatic components of the ISG15 conjugation cascade (Fig. 3b). These cells thus would express the ISG15 conjugation system upon Dox treatment independently of IFN. Fig. 3c shows that one of the isolated clones (U251.ISG7-8) showed evidence of significant induction of ISGylation under Dox treatment. We then sought to determine whether ISGylation affected proteins associated with p62 in soluble versus insoluble cellular subfractions. Uninduced or Dox-induced cells were treated with pepstatin A and E64d (PepA/E64d; inhibitors of lysosomal proteases), and then soluble and insoluble fractions of cell lysates were analyzed by Western blotting. Fig. 3d shows that Dox-treatment induced ISGylation of the proteome in both soluble and insoluble fractions and that this was even more evident when cells were treated with PepA/E64d. Interestingly, Dox treatment resulted in perhaps a mild increase in p62 levels in the soluble fraction, whereas LC3-II levels were increased a bit more convincingly.   Lysates were subjected to co-immunoprecipitation (IP) with antibody against FLAG and analysis using antibodies against FLAG and ISG15. c, 293 cells were transiently co-transfected with expression vectors for FLAG-HDAC6 WT, a C-terminal truncation (⌬Cter) mutant, an N-terminal truncation (⌬CD1/2) mutant, a BUZ domain truncation (⌬BUZ) mutant, an SE14 domain truncation (⌬SE14) mutant, or an empty vector (Vec) along with an HA-tagged ISG15 expression vector. Lysates and immunoprecipitates were analyzed with antibodies against FLAG and HA. d, schematic of HA-tagged ISG15 with wild type or mutants. The two Ub-like domains are shown as closed boxes. Peptides were tagged with HA at the N terminus, and amino acids were deleted from the LRLRGG peptide sequences at the C-terminal end (c-end) as indicated by asterisks (*). e, 293A cells were transiently transfected with plasmids carrying HA-tagged ISG15 vectors with mutants. In a pulldown assay, cell lysates were incubated with purified recombinant GST-tagged HDAC6 or control GST, bound to glutathione-Sepharose beads, and subjected to analysis with antibodies against HA and GST. IB, immunoblotting.
However, there was a clear increase by Dox of p62 and LC3-II in the insoluble fraction that was even more visible in PepA/E64dtreated cells. Similar findings were also obtained in IFN␤treated U251 cells (see Fig. 6). These results suggested that induction of ISG15 and ISGylation leads to up-regulation of p62 and LC3-II association with IGS15-conjugated proteins ending up in the lysosomes under condition of both Dox-induced ISGylation and IFN stimulation (from Fig. 6). The association with p62 and LC3-II strongly suggests an autophagic mechanism for degradation of ISG15.

ISG15 Associates with HDAC6 and p62
To analyze the in vivo interaction between p62 and ISG15conjugated proteins, we stably transduced 293 cells to express the ISG15 conjugation system in response to Dox (293.ISG8 cells) as shown in Fig. 3e. These 293.ISG8 cells were then transiently transfected with human p62 containing a C-terminal FLAG epitope tag (FLAG-p62) or empty vector. Fig. 3f shows that FLAG-p62 co-immunoprecipitated with ISG15-conjugated substrates. Interestingly, MG132 led to a visible decrease in FLAG-p62 levels in the input lysates that were extracted from the detergent-containing lysis buffer (Fig. 3f). To further try to understand this, we decided to analyze the detergentinsoluble fraction for p62 levels as a function of MG132 doses with and without Dox treatment. Fig. 3g shows that, in the absence of Dox, p62 was present primarily in the soluble fraction without an effect from proteasome inhibition. With Dox (thus increasing ISGylation activity) and increased doses of MG132, there was a visible shift of p62 from the soluble to insoluble fractions. Because MG132 treatment blocks the autophagic degradation pathway of aggregates, there was also an MG132 dose-dependent increase in LC3, ISGylated proteins, and HDAC6 in the insoluble fraction, although a lot of these were still present in the soluble fraction. These findings thus suggested that ISGylation led to a shift of p62, HDAC6, and LC3 into the insoluble aggregate fraction of cells that was detectable once the proteasome was inhibited.
Therefore, to determine whether p62 interacted and directed ISG15 to accumulate in inclusion bodies in response to IFN, we prepared prepermeabilized IFN-treated U251 cells and immu-nostained them using antibodies against p62, ISG15, and ␣-tubulin (Fig. 4). In the absence of MG132, cells contained numerous small punctate areas of staining for p62 bodies in the cytosol, most of which also co-localized with ISG15 (Fig. 4a). The p62-positive puncta became visibly larger in MG132treated cells, and this was also true for puncta in which ISG15 and p62 co-localized (Fig. 4b). ISG15 puncta were also observed on detergent-resistant stable microtubules regardless of MG132 treatment (Fig. 4, c and d) that accumulated ISG15positive large aggregates at the microtubule-organizing center (Fig. 4d). These results suggested that p62 contributes to the formation of ISG15-containing aggregates in the microtubuleorganizing center.
An Engineered ISG15 Protein Is a Target of Lysosomal Degradation-We also attempted to detect ISG15 conjugate degradation using an engineered ISG15 conjugate. For this, we engineered a CFP-fused ISG15 expression vector in which the C-terminal LRLRGG is linked to the N terminus of mTur-quoise2 (CFP) protein (Fig. 5a). As shown in Fig. 5b, we could not detect the fusion protein upon DNA transfection in the absence of interferon but instead observed separate immunopositive bands for ISG15 and mTurquoise2 proteins, suggesting fairly prompt cleavage of the expressed fusion protein after transfection into cells. This observation appears to agree with that describing precursor processing of ISG15 to expose LRLRGG-terminal peptides by proteases (30). We then took advantage of this observed cleavage of ISG15-CFP protein to evaluate its lysosomal degradation (Fig. 5c). Similar to the  results of Fig. 3, cleaved ISG15 protein was observed in the detergent-insoluble fraction, and PepA/E64d treatment caused accumulation of the ISG15 protein. These findings thus suggested that ISG15 protein can be a target of lysosomal degradation.
The Deacetylase Activity of HDAC6 Contributes to ISG15 Degradation-We showed in Fig. 2 that the BUZ domain of HDAC6 was essential for binding to ISG15 protein, and it has also been shown that the deacetylase activity of HDAC6 is involved in the degradation of the aggresome, which contains ubiquitinated proteins, via autophagy-lysosome degradation (10). In a similar fashion, we thus sought to determine whether HDAC6 activity was involved in ISG15 degradation. Fig. 6 shows that type I IFN as expected visibly increased ISG15 levels and also mildly increased ISG15 conjugates in both soluble and insoluble fractions. When PepA/E64D lysosomal protease inhibitors were added, there was a visible increase in ISG15 levels in the insoluble fraction. This suggests that ISG15 can be degraded by lysosomal proteases.
We then utilized tubacin (20), a relatively selective HDAC6 inhibitor, and observed a visible decrease of ISG15 levels in both soluble and insoluble fractions without much of a change in ISG15 conjugate levels. However, when PepA/E64D lysosomal protease inhibitors were also added, there was no change in ISG15 levels. This suggested that HDAC6 was needed for the degradation of ISG15 by lysosomal proteases. Tubacin also led to an increase of p62 and LC3 levels in the insoluble fraction. Therefore, inhibition of HDAC6 activity led to the accumulation of p62 and LC3-II in the insoluble fraction, suggesting inhibition of lysosome-autophagosome fusion. It also reduced total ISG15 levels in cells in response to type I IFN, but this did not lead to an increase in ISG15 when lysosomal proteases were inhibited. Those results suggested that the deacetylase activity of HDAC6 is important for degradation of ISG15 via the lysosome.

DISCUSSION
The ubiquitin-like post-translational modifier ISG15 is upregulated during type I IFN stimulation of cells, and it becomes conjugated to newly synthesized proteins of the host cell and invading pathogens. Although this conjugation can be reversed by a specific protease, UBP43/USP18 (3), it is not known whether other cellular pathways may also be operative. In this report, for the first time, we show that 1) ISG15 interacts via its LRLRGG C-terminal motif with a BUZ domain of cytosolic Ub-binding HDAC6, implicating HDAC6 for a relevant role in the function of ISG15. That this interaction is the same as that known to occur for (poly-) ubiquitin chains binding to HDAC6 also suggests that the ISG15 pathway may represent the IFNstimulated counterpart to the normal homeostatic ubiquitin clearance pathway. 2) ISG15 also interacts with the cargo adapter protein SQSTM1/p62. The knowledge that both p62 and HDAC6 facilitate aggregate formation (aggresomes) and clearance of ubiquitinated cargo through the LC3-II-marked autophagosome and lysosome also suggested that a clearance process (similar to that of Ubiquitination) may be occurring with ISGylation of proteins during the innate immune response. Therefore, our data strongly suggest that ISG15 conjugation marks target substrates for clearance via autophagylysosome pathway as proposed in the model shown in Fig. 7.
We should note that the reported data were mostly obtained under experimental conditions of forced overexpression, and we have not been able to detect this under normal, homeostatic physiological conditions. This implies that the observed results may occur primarily when the cell is stressed (forced overexpression of cDNAs, interferon stimulation, and inhibition of the proteasome and lysosomal proteases).
The structural conformation of ISG15 resembles that of Lys 63 -linked di-Ub rather than that of Lys 48 -linked di-Ub, which is known to be associated with the proteasomal pathway.
Lys 63 linkage appears to function in homeostatic cellular processes including signal transduction (31). In addition, when molecular chaperones and proteasome degradation systems are unable to unfold and thread misfolded/aggregated proteins through the narrow interior proteolytic chamber of the 26 S proteasome, these proteins get tagged with Lys 63 , designating them for the autophagy-lysosome pathway (11,13). Molecularly, this happens through p62, which preferentially binds to polyubiquitinated proteins with Lys 63 -linked chains instead of those with Lys 48 -linked chains, resulting in the formation of oligomeric aggregates of ubiquitinated proteins, organelles, and intracellular pathogens that lead to autophagic rather than proteasomal degradation (15,25,32,33). This is also in agreement with evidence indicating that Lys 63 -linked polyubiquitin substrates are enriched in intracellular inclusion bodies, which are also sequestered into p62-LC3-marked autophagosomes (34). Results in the present study show that p62 also binds to non-covalent ISG15 by pulldown assay and to ISG15-conjugated proteins by co-immunoprecipitation assay. In addition, using a ISG15 fusion protein with CFP via linkage of LRLRGG, we clearly showed that ISG15 itself is a target of lysosomal degradation (Fig. 5c). Therefore, this suggests that p62 also directly targets ISG15 and ISGylated peptides to form aggregates and autophagosomes in a manner that is similar to p62 interaction with Lys 63 -linked polyubiquitinated proteins being targeted to autophagosomes.
ISG15 and its conjugates have been shown to possess activity against several viruses using cultured cells or animal models (4). c. d.

PepA/E64d
Tubacin MG132 FIGURE 7. Schematic model of ISG15-mediated degradation. a, IFNs are known to stimulate the JAK-STAT pathway to induce a number of interferonstimulated genes including ISG15, UbE1L, UbcH8, and Herc5 that lead to the ISGylation process and UBP43 that deconjugates ISGylated proteins. b, the Lys 63 -linked di-Ub-like protein ISG15 conjugates to lysine (K) residues of newly synthesized proteins at the LRLRGG-terminal end (2). Some ISG15 conjugates are also deconjugated to remove ISG15. c, our findings indicate that HDAC6 and SQSTM1/p62 can independently bind ISG15 to recognize ISG15 and ISG15 conjugates directly or indirectly. ISG15 aggregates or aggresomes would be formed through oligomerization and recruitment of autophagosome-bound LC3 by p62. The proteasome inhibitor MG132 induces and enhances this aggregate formation and accumulation in the insoluble fraction. d, the deacetylase activity of HDAC6 can mediate lysosomal fusion of ISG15-containing aggregates for clearance, and this is inhibited by the specific HDAC6 inhibitor tubacin. PepA/E64d inhibit lysosomal digestion of peptide contents.
The antiviral mechanisms of ISG15 include inhibition of viral release and budding as well as direct ISG15 conjugation of cellular proteins including antiviral factors (e.g. IRF3, retinoic acid induced gene-I (RIG-I), and protein kinase R) and other ISG proteins. Also, ISG15 can be directly conjugated with capsid proteins of human papillomavirus and Sindbis virus (2,35). However, it is unknown how ISG15 modification of viral proteins acts mechanistically. Our findings show that ISG15 not only co-localizes with p62 and HDAC6 in the same intracellular punctate areas (puncta) but also associates with them. p62 and other sequestrome-1-like receptors function as cargo adaptor proteins that target substrates to autophagosomes via the LC3interacting region motif. In xenophagy, ubiquitination of invading bacteria is required for antimicrobial autophagy through p62 cargo recognition and degradation (36). In a similar manner to antiviral autophagy (virophagy), capsids of Sindbis virus and Chikungunya virus are targets of autophagosomes through binding of p62, and Chikungunya virus capsids are also modified by ubiquitin (37,38). Therefore, future studies should elucidate whether ISG15 enhances degradation or sequestration of viral proteins via p62-and HDAC6-mediated selective autophagy.
HDAC6 functions to deacetylate cytoplasmic proteins (e.g. ␣-tubulin), and catalytic inhibition leads to hyperacetylation of ␣-tubulin, which stimulates canonical (bulk) autophagy by kinesin-1 recruitment to microtubules for autophagosome transport toward the microtubule-organizing center (39,40). In non-canonical (selective) autophagy, HDAC6 regulates the fusion of autophagosome and lysosome through both catalytic deacetylase and ubiquitin binding activities (10). We also observed that this deacetylase activity is associated with ISG15 degradation via the lysosome-autophagosome (Fig. 6). HDAC6 preferentially interacts with Lys 63 -linked polyubiquitinated protein aggregates in vivo (16). However, there is no biochemical evidence that HDAC6 directly binds to Ub-conjugated proteins in vitro (12). We also identify for the first time in this report that the BUZ domain of HDAC6 interacts with the C-terminal LRLRGG peptide of free ISG15. Coupled with the co-immunoprecipitation evidence of FLAG-HDAC6 interaction with ISG15 conjugates, we speculate that HDAC6 may recognize and associate with unanchored free ISG15 in the p62 cargo, which also sequesters the substrates of ISG15 conjugation.
The multifaceted adapter p62 plays critical roles in the regulation of a broad range of signaling pathways that respond to inflammatory and oxidative stresses through its binding to Lys 63 -linked Ub proteins and related binding partners such as tumor necrosis factor receptor-associated factor 6 and Kelchlike ECH-associated protein 1 (31,41). ISG15 is also involved in the innate immune signaling pathway. In fact, IFN-induced ISG proteins including IRF3 are known substrates of ISG15 conjugation, and their function is modulated by ISGylation (42). Our study also shows for the first time that p62 is involved in ISG15mediated signaling pathways in a manner similar to that of p62 involvement in Lys 63 -linked Ub-mediated signaling in published reports (15,25). However, it is not known how p62 distinguishes ISG15-conjugated proteins that remain active to perform a function from those that are targets for a degradation tag. One possible explanation is that, for ISGylated proteins to remain active, p62 scaffolding with ISG15 conjugates forms without free ISG15 and/or free Lys 63 -linked Ub chains. Conversely, for proteins destined for degradation, sequestration into disorganized aggresomes occurs (and this could be induced by blocking the UPS with MG132) in combination with HDAC6 and LC3 recruitment (12). In fact, we did find accumulation of free ISG15 in the detergent-insoluble fraction of cells with p62 and lipid-bound LC3-II as a function of MG132 dose. Furthermore, the catalytic inhibition of HDAC6 with tubacin also suppressed lysosomal ISG15 degradation. Therefore, it is likely that type I IFN signaling induces ISG15, which in turn strengthens the basal activity of selective autophagy, leading to efficient removal of ubiquitin and ISG15-tagged unwelcome proteins and pathogens through p62 and HDAC6.
In summary, our studies provide novel evidence for ISG15 association with HDAC6 and p62, implying that ISGylation can lead to autophagic degradation in pathways that are similar to those utilized by the Ub system. ISG15-mediated protein conjugation may thus be the IFN-based system that, together with or in lieu of Ub-based selective autophagy, allows the cell to use autophagy as an intrinsic antipathogen defense.


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Computer Science
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Intrinsic electrical properties of mammalian neurons and CNS function: a historical perspective

This brief review summarizes work done in mammalian neuroscience concerning the intrinsic electrophysiological properties of four neuronal types; Cerebellar Purkinje cells, inferior olivary cells, thalamic cells, and some cortical interneurons. It is a personal perspective addressing an interesting time in neuroscience when the reflex view of brain function, as the paradigm to understand global neuroscience, began to be modified toward one in which sensory input modulates rather than dictates brain function. The perspective of the paper is not a comprehensive description of the intrinsic electrical properties of all nerve cells but rather addresses a set of cell types that provide indicative examples of mechanisms that modulate brain function.

INTRODUCTION
That the function of the nervous system is ultimately to be defined as the product of interacting networks woven by nerve cells has been the central dogma of neuroscience for almost a century. Fundamental to this view has been the realization that nerve cells are truly individual elements. Indeed, while the variety of forms that nerve cells may display was described in elegant detail by the work of brilliant morphologists of the turn of the century, their most significant contribution was the proposal of the neuron doctrine (cf. Ramón y Cajal, 1904).
On the other hand, from a physiological point of view the neuron doctrine was considered for a long time to signify a unity of excitability, where the variance among the different neurons related to their shape and connectivity, but not to their individual electrophysiological properties. Thus, following the discovery of excitatory and inhibitory synaptic potentials, it was assumed that the necessary and sufficient functional coinage for the expression of functionality in nerve nets had been defined. Over the past 30 years, however, another fundamental issue has arisen with respect to the physiological properties of nerve cells-that of their intrinsic electroresponsive properties. This concept may be stated simply: "Neuronal types are not interchangeable." That is, a neuron of a given type (e.g., a thalamic cell) cannot be functionally replaced by one of another type (e.g., an inferior olivary cell), even if their synaptic connectivity and the type of neurotransmitter outputs are identical. (The difference is that the intrinsic electrophysiological properties of thalamic cells are extraordinarily different from those of inferior olivary neurons).
This being the case, the intrinsic electrophysiological signature of nerve cells becomes a central theme in neuronal function. Indeed, when such elements interconnect, the dynamics of the resulting neuronal networks are governed not only by the flow of synaptic current, but also by the intrinsic properties of the neurons partaking in such circuits. Likewise, the electrical activity observed in a network is not only related to the excitatory and inhibitory interactions among neurons but also to their inherent or intrinsic electrical activity (Llinás and Hess, 1976;Llinás, 1988).
The term "intrinsic electrical properties" has been used to encompass both passive and active membrane characteristics (for example, see van Lunteren and Dick, 1992). In this review it is used in a more restricted sense to designate those active membrane properties that endow a cell with the ability to shape incoming stimuli and indeed to fire or maintain subthreshold oscillations in the absence of synaptic input. That is, these cells are capable of more than the classical input-output relationship of increasing their firing frequency with stimulus strength or action of neuromodulators (see Binder et al., 1993, for motoneurons). Due to the presence of bursting, neurons with such usual properties were first recognized in systems concerned with rhythmic activity such as breathing, swallowing and chewing. Indeed, the rhythmic firing of hypoglossal neurons was reported as early as 1973 (Lund and Dellow, 1973). However, the contribution of intrinsic electrical properties of hypoglossal motoneurons to such periodicity was not recognized, but rather thought to arise from the action of excitatory and inhibitory synaptic inputs, the presence of gap junctions, and input from a central pattern generator. It was not until much later that the role of the intrinsic electrical properties of the neurons themselves was recognized (see Ramirez and Richter, 1996, for a review of respiratory neurons). In fact, when hippocampal neurons were observed to fire spontaneously when inhibitory input was blocked, the authors concluded "It remains to be determined whether neural properties and connectivity found to be important in this hippocampal rhythm may also play a role in the generation of other rhythmic activities in the mammalian CNS" (Wong et al., 1984). Such spontaneous rhythmicity had been reported for the inferior olive (IO) in vivo as early as 1968 (Armstrong et al., 1968). That they were indeed intrinsic to the IO cell was shown in 1986 (Llinás and Yarom, 1986).
Below, some examples are given from our work that illustrate different intrinsic electrical properties in mammalian central neurons. Very surprising, the role of intrinsic activity in the generation of motricity, initially proposed by Graham-Brown (1911), was forgotten for more than halve a century.

INTRINSIC ELECTRICAL PROPERTIES OF SPECIFIC CELL TYPES CEREBELLAR PURKINJE CELLS
The question of intrinsic electroresponsive properties in vertebrate CNS neurons was first encountered in the detailed study of cerebellar Purkinje cells (Llinás and Hess, 1976;Llinás and Sugimori, 1980a,b). These studies demonstrated that Purkinje cells have intricate firing properties and that the dendritic and somatic membranes each have markedly different voltagedependent conductances that are supported by different types of ionic channels, which combine to give these cells their unique firing signature. At the somatic level, in addition to the Na + and K + conductances that generate the fast action potential, a voltagedependent, persistent, or very slowly inactivating Na + conductance [g Na(p) (p for persistent)] was also initially encountered in these neurons (Llinás and Sugimori, 1980a). This latter conductance generates a slow, tetrodotoxin (TTX)-sensitive depolarizing response, which, once activated generates prolonged plateau potentials that may last for tens to hundreds of milliseconds (presently known as an "up state").
This gNa(p) has also been described in cortical (Connors et al., 1982;Stafstrom et al., 1982) and thalamic (Jahnsen and Llinás, 1984a,b) neurons. The dendrites of Purkinje cells, by contrast, do not support voltage-gated Na + conductances, but rather voltage-gated Ca 2+ conductances that generate dendritic Ca 2+dependent spikes and/or plateau potentials (Llinás and Hess, 1976;Llinás and Sugimori, 1980a,b) and are supported by a calcium channel named the P channel (for Purkinje cell). These different membrane conductances and their distribution over the somato-dendritic plasmalemmal membrane endow Purkinje cells with intricate electroresponsive properties, including intrinsic transmembrane voltage oscillations. Such activity can be evoked by direct current injection or by extracellular iontophoretic application of an excitatory transmitter such as glutamic acid at the dendritic level (Figure 1).
The characteristics of somatic and dendritic electroresponsiveness to dendritic glutamate application have been studied using double impalement of Purkinje cells in cerebellar slices. Recordings made during one such experiment in which glutamic acid was applied to the distal dendritic tree are shown in Figure 1.
The schematic to the left shows the approximate location of the dendritic and somatic recording electrodes and the iontophoretic glutamate electrode. The trace in B illustrates the main features of dendritic electroresponsiveness. There are two types of Ca 2+ -dependent responses: maintained all-or-none depolarizing plateau responses and slow-rising spikes. The plateau responses Intradendritic recording. The large amplitude wide action potentials are Ca-dependent while the smaller fast action potentials represent the passive invasion of the somatic action potentials into the dendritic tree. Note the presence of a sustained plateau depolarization at the dendritic level following the spiking phase of the dendritic response. (C) Simultaneous intrasomatic recording showing fast somatic Na-dependent action potentials. Note that each of the large somatic spikes is seen at dendritic level with a short delay and that the calcium dependent dendritic spikes generate high frequency spiking as somatic level. (D) Superposition of dendritic (red) and somatic (blue) spikes to illustrate the temporal relationship between somatic and dendritic spikes and plateau amplitudes (Llinás and Sugimori, 1980a,b. This example is unpublished).
have constant amplitude, may last for hundreds of milliseconds, are accompanied by a large conductance increase, and are usually not seen in the soma. On the other hand, the Ca 2+ -dependent spikes in the dendrites are large and are usually elicited in prolonged bursts (Figure 1B), which influence somatic electroresponsiveness. As shown in Figure 1C, they may be recorded in the soma as slow changes in the membrane potential that trigger increases in the firing frequency of the fast, sodium-dependent somatic action potentials. In turn, the somatic action potentials can be observed in the dendrites below the mid-dendritic level as small, fast-rising depolarization.
The ionic basis for Purkinje cell firing was examined by studying the response to depolarizing pulses in the absence of Ca currents and in the absence of Na currents (Figure 2). Addition of Co to the bath blocked the calcium conductance. Direct depolarization elicited fast somatic spikes on a slow depolarizing ramp bringing the membrane to a plateau potential. When the amplitude of the depolarizing pulse was increased the depolarizing ramp occurred earlier ( Figure 2B, arrows) without changing the spike threshold or plateau level ( Figure 2B). Pharmacological block of the fast sodium channel by addition of TTX to the bath changed the firing pattern as shown in Figure 2D. The fast somatic spikes were blocked while the slow dendritic spike burst and afterdepolarization (Figure 2D, arrow) remained (compare Figure 2C and Figure 2D). These experiments have provided valuable information relating to the ionic basis of the electrical responsiveness of the soma and dendritic trees and allowed the determination of electrotonic length and some of the active membrane properties of Purkinje cells in general. Yet, they do not provide a direct demonstration of the spatio-temporal distribution of electroresponsiveness over the entire soma dendritic membrane. This requires the use of techniques such as ion-sensitive dyes.
The spatial distribution of ionic channels over the plasmalemmal membrane and the associated compartmentalization of both the physiological and the cell biological properties are critical issues in the characterization of central neuronal function. For example, the precise distribution of specific channels with respect to the locus of synaptic input may address not only electrical integrative properties but also the precision with which different compartments may be addressed biochemically. Indeed, the spatial distribution of second messenger systems activated by [Ca 2+ ] i (Hemmings et al., 1986) will be determined by the distribution of calcium channels.
An early experiment of this type was carried out 26 years ago by Tank et al. (1988). In these experiment Fura II signals were used to determine [Ca 2+ ] i in Purkinje cells (Tank et al., 1988). Fura II was injected ionophoretically into the cell and a quantitative evaluation of changes in [Ca 2+ ] i was made using the fluorescence ration at 340/380 nM as seen in Figure 3.
The results agreed with the hypotheses of the dendritic segregation of Ca 2+ conductances that was suggested by early electrophysiological experiments (Llinás and Hess, 1976;Llinás and Sugimori, 1980a,b). They also allow a general mapping of the location of voltage-gated Ca2+ channels as inferred from the specific regions of the neuron, where [Ca 2+ ] i demonstrate transients lasting 5-15 ms.  (From Tank et al., 1988.).
With respect to the functional significance of the results, these findings indicated that the Ca 2+ -dependent plateau potentials are a dendritic boosting mechanism for the synaptic current generated in Purkinje cell dendrites leading to a high-frequency burst of sodium spikes at the soma and axon. This provides a mechanism for spatial and temporal summation of inhibition at the cerebellar nuclear neurons. Other possibilities to be considered relate to role in increased intracellular calcium in the modulation of cell biological mechanisms and the modification of long-term cell biological properties.
The next approach in Purkinje cells was to carry out direct single channel recordings (Figure 4) at both the somatic and dendritic levels (Usowicz et al., 1992). The location of channels on dendrites made it clear that, given the dendritic surface to volume factor relationship, calcium dye imaging would be more effectively implemented at dendritic level. On the other hand, it was also clear that the final calcium concentration change at the cytosolic level would be larger and probably longer lasting at the dendritic than at the somatic level.
In short then, the evidence was clear that Purkinje cells have complex intrinsic properties from the merging of dendritic and somatic conductances giving these cells a unique electrophysiological signature.

INFERIOR OLIVARY CELLS AND REBOUND CALCIUM SPIKES
Cells of the inferior olivary nucleus have also been shown to have dendritic and somatic conductances underlying an intrinsic electrophysiological profile. Indeed, in vitro experiments using brainstem slices (Llinás andYarom, 1981, 1986) first demonstrated that IO neurons have a set of voltage-gated ionic conductances that give these cells intrinsic oscillatory properties ( Figure 5). Thus, the firing of IO cells is characterized by an initial fast-rising action potential (a somatic sodium spike), which is prolonged to 10-15 ms by an afterdepolarization (a Ca 2+ -dependent dendritic spike). The abrupt long-lasting afterhyperpolarization (AHP) following the plateau afterdepolarization totally silences the spikegenerating activity. This hyperpolarization is typically terminated by a sharp, active rebound response ( Figure 5A, arrow), which arises when the membrane potential is negative to the resting level. This rebound response is due to the activation of a somatic Ca 2+ -dependent action potential and results from a second voltage-dependent Ca 2+ conductance, which is inactive at the resting membrane potential (−65 mV). Membrane hyperpolarization deinactivates this conductance, and, as the membrane potential returns to baseline, a "low threshold" Ca 2+ -dependent spike is generated (Llinás and Yarom, 1981). The rebound potential can be modulated by small changes in the resting membrane potential such that a full Na + spike, which, in turn, can set forth the whole sequence of events once again, is activated. In this way, the cell will fire at a frequency determined largely by the characteristics of the AHP ( Figure 5B).
A direct demonstration of time course and amplitude of the "low threshold" transient calcium current [ICA (T)], encountered in this neuron (Llinás and Yarom, 1981) is shown in Figure 6, following a voltage clamp study of IO neuronal calcium currents.
The low threshold, transient calcium current is a powerful modulator of IO rhythmicity and is responsible for IO membrane potential oscillations. IO neuron oscillations can occur at two distinct frequencies, as determined by examining the firing properties of spontaneous bursts of spikes. A set of such events is shown in Figure 7.
It is evident, given the above, that individual IO cells can oscillate with two main limit cycles, one near 10 Hz (9-12 HZ) and the other near 4 HZ (3-6 HZ). Oscillation at the higher frequency seems to be governed by the resting potential of the neuron. Thus, when the cell is depolarized, its excitability would be dominated by the dendritic conductances and fire near 4 Hz. However, when the cell is hyperpolarized, its output is dependent on somatic conductances and will fire near 10 Hz.
Beyond its intrinsic oscillatory behavior, one of the most unexpected and novel properties of the dynamics of IO neurons was their phase-reset ability. Thus, synaptic input large enough to activate action potentials also produces a phase reset of the  Table giving the distribution of ionic conductances in somatic and dendritic regions. At the soma a set of conductances (g Na and g k ) generating fast action potentials may be observed. In addition, a strongly inactivated Ca 2+ conductance is present, which produces rebound spikes, as seen in (B) [g Ca (somatic) ]. Also recorded at the soma is a large Ca 2+ -dependent dendritic spike [g Ca (dendtritic) ] that generates the afterdepolarization and the powerful, long-lasting afterhypolarization, which is produced by a Ca 2+ -dependent K+ conductance [g K (Ca) ]. In addition, a voltage-dependent K + conductance (g K ) seems to be present in the dendrites. Right A: Rebound spikes in the inferior olivary neuron (arrow) following blockage of the Na spike with tetrodotoxin (TTX). Right B: Summary of the ionic conductances that generate single-cell oscillations in neurons of the inferior olive. oscillatory rhythm that is independent of the phase point at which the stimulus arrived. As shown in Figure 8A, a stimulus large enough to generate a spike discharge is immediately followed by a rapid return of the membrane potential oscillatory behavior. If the spike-activating stimulus is repeated, as in Figure 8B, it becomes apparent that the resultant oscillatory phase reset is the same regardless of the moment in time when the stimulus was delivered. These oscillatory membrane potential properties can also be demonstrated to have interesting dynamic properties. Analysis of the oscillatory dynamics such as shown in Figures 8A,B demonstrated that IO cells have attractor properties as reconstructed from a time series analysis that has a structure close to a limit cycle with a regular periodic trajectory (Makarenko and Llinas, 1998). Average mutual information and false near neighbor methods were calculated and are shown in Figures 8C,D.
To reconstruct the attractor and Lyapunov exponents were derived and the results demonstrate zero, positive and negative exponents values indicating that the system displays lowdimensional chaotic dynamics, that actually explain the phase rest properties of their oscillation, as shown in Figure 8B. The application of this modeling to IO dynamics shown that the subthreshold oscillations support low dimensional chaotic dynamics and that IO electronic coupling leads to rapidly generated complex functional states without increasing the dimensionality of the system (Makarenko and Llinas, 1998).
Thus, in addition to uniform membrane potential oscillatory properties, because of their dynamics IO neurons have the unique ability to reset their oscillatory phase when activated (Leznik et al., 2002;Lefler et al., 2013). This reset property has been found to be functionally very significant as it allows a rapid reset of motricity when dominated by the somatic conductances and fire near 10 Hz-the basic rhythmicity of motor control in vertebrates (Vallbo and Wessberg, 1993;Lang et al., 2006). It has also been shown to be essential in the rapid reorganization of motricity following motor stumbling, even under robotic control (Porras and Llinás, 2014).
At the cerebellar level the functional significance of the oscillatory properties illustrated in Figure 8 is an increased probability of Purkinje cell complex spike activation relating to rapid recovery of motor execution following stumbling, or other unpredicted motor events. Ultimately, then IO oscillatory activity is required for proper motor execution, as demonstrated by the total ataxia that follows T type calcium channels knockout (Choi et al., 2010).

THALAMIC CELLS
Thalamic neurons also have complex intrinsic properties that allow them to function either as relay systems, or as oscillators and/or resonators. That these two modes are intrinsic to the cells and are controlled by their membrane potential has been studied both in vitro (Llinás and Jahnsen, 1982;Jahnsen and Llinás, 1984a,b;Hirsch et al., 1985;Prince, 1986, 1987;Crunelli et al., 1987;Wilcox et al., 1988) and in vivo (Deschenes et al., 1984;cf. Steriade and Llinás, 1988) The basic electrophysiological phenomenology observed in these cells is shown in Figure 9.
From a slightly depolarized membrane potential, the outward current injection elicited a subthreshold depolarization ( Figure 9A, second trace). When the current pulse was delivered from a more depolarized potential, regular, tonic firing was elicited as shown in the top trace of Figure 9A. Thus, at or near the resting potential, tonic firing is elicited by membrane depolarization. Accordingly, the response to an excitatory synaptic input would be a single excitatory postsynaptic potential (EPSP) that may trigger single spikes. A very different response was elicited when a similar current pulse was delivered from a hyperpolarized level as in Figure 9B. Under these conditions, the same outward current pulse showed in A, triggered an all-ornone burst of spikes. The uniformity of the waveform of the burst is demonstrated by the fact that several traces are superimposed in Figure 9B. The response comprises two distinct parts, a low-threshold spike (LTS), a slowly rising and falling triangularlike potential, and a rapid succession of fast spokes. As in the IO, the LTS is due to activation of a Ca 2+ conductance that is deinactivated by membrane hyperpolarization. The amplitude of the low-threshold response is related to the membrane potential before its generation. This is shown in Figure 9C where a series of hyperpolarizing pulses of increasing amplitude was delivered from a slightly hyperpolarized membrane level. The rate of rise and amplitude of the rebound response elicited at the current break increased with progressively larger hyperpolarizing pulses. At the two highest levels, the rebound potential reached the firing threshold for Na + -dependent spikes. The deinactivation of the low-threshold Ca 2+ -dependent spike is also time-dependent; hyperpolarizing pulses of increasing duration produce graded deinactivation (Jahnsen and Llinás, 1984a), and complete recovery of the LTS occurs after a refractory period of 170-200 ms. Another characteristic of thalamic neurons, the presence of an A-like potassium current, may also be seen in Figure 9C as a longer time course to repolarization. Deinactivation of this conductance is responsible for the delay in the return of the potential to the holding potential at the end of the current injection.
A direct demonstration of the low threshold calcium conductance can best be described by the results from thalamic neuron voltage clamping. Such results are illustrated in Figures 9D,E. Indeed, the time course of the calcium inactivating T current is clearly demonstrable from a holding potential of 75 mV following Na and K conductance block by TTX and TEA respectively. The activation property of this current is shown in graph Figure 9E.
In addition to these voltage dependent conductances, thalamic neurons can modify their synaptic properties depending on membrane potential in a quite remarkable fashion. These properties are often not taken into account when considering their effects on arousal. Thus, fast reversible synaptic plasticity occurs in the thalamus by changes in postsynaptic membrane potential, independently of presynaptic volley size, and is rapidly reversible. It represents one of the few examples of rapid postsynaptically dependent synaptic plasticity as illustrated in Figure 10.
The significance of such finding resides in the fact that the thalamocortical system can quickly select functional states relating to gamma band allowing cognitive attractors to be continuously modulated by the combination of recurrent thalamocortical activity and the sensory input from the external world.

THALAMIC 40 Hz OSCILLATIONS
In addition to the now well-known thalamic currents responsible for the wake-sleep cycle (Steriade and Llinás, 1988), in vitro studies indicate that, in addition to the low frequency and alpha rhythms, a gamma band rhythm is also present in thalamic neurons. This is particularly clear at dendritic levels and is supported by P/Q type calcium channels (Pedroarena and Llinás, 1997) and is essential in the generation of cognitive functions (Llinás et al., 2007). Indeed depolarization by direct current injection elicits well defined high frequency at potentials of −46 and −43 mV ( Figure 11A) and the oscillations that can reach threshold for spike initiation at −40 mV ( Figure 11B). These high frequency oscillations were blocked by P/Q channel blocker SFtx Mintz et al., 1992;cf Nimmrich and Gross, 2012). The relationship of dendritic spikes and gamma oscillations was examined in a mathematical model of thalamocortical relay cells (Rhodes and Llinás, 2005). The model incorporated the generation of somatic spikes, low threshold rebound spike bursts, and fast somatic oscillations near threshold. In the distal dendrites the model neuron generated both isolated high-threshold calcium spikes and low threshold calcium spikes that did not require a high dendritic density of calcium channels. Somatic depolarization elicited firing in a dendrite (Figure 11C, red electrode; Figure 11D, red traces) that leads to subthreshold oscillations in the some (Figure 11D, white trace). When somatic depolarization ended, arrows in Figure 11C, dendritic spiking stopped. A similar pattern was seen when the location of the dendritic electrode was moved (blue and green in Figures 11C,D). The period of firing in the distal dendrites controlled the somatic oscillation frequency.
The gamma oscillations are generated in the dendrites as shown by dye imaging studies such as shown in Figure 11E where fluorescence of the calcium-specific dye flura 2 is restricted to the dendrites (Pedroarena and Llinás, 1997).
In thalamo-cortical slices, where the reciprocal connectivity is intact, thalamic stimulation results in the recurrent activity of the thalamocortical loop ( Figure 11F). It is interesting to note that the high frequency cortical return excitation is mostly restricted to the dendritic thalamic compartment ( Figure 11E). From the above it has been concluded that this dendritic conductance are not only related to oscillatory gamma band activity, but are essential in the generation of brain gamma band activity and of cognitive functions, as was demonstrated in mice genetically modified to delete P/Q type channels (Cav 2.1 null mice) (Llinás et al., 2007).
In all then, six ionic conductances have been described in thalamic neurons, in addition to those that underlie the axosomatic action potential as summarized in Figure 12.
(1) A voltage-dependent persistent, or very slowly inactivating somatic, Na + conductance, [g Na(Ip) ] This conductance generates a slow rebound depolarization in thalamic cells and plays a role in the genesis of the 10-Hz oscillation Figure 10 (Jahnsen and Llinás, 1984b).
which underlies the AHP. This conductance was demonstrated in vitro by the marked reduction of the AHP, after application of Ca 2+ channel blockers (Jahnsen and Llinás, 1984b). The AHP amplitude is about 12 mV and can be reversed by inward current injection (Deschenes et al., 1984;Jahnsen and Llinás, 1984b). (3) A fast, transient somatic potassium conductance (g IA ), responsible for the slow return to baseline following hyperpolarization (Jahnsen and Llinás, 1984b;Kita and Kitai, 1986), which can prevent the abrupt recovery of neurons from a hyperpolarized condition. In fact, the duration of the hyperpolarization that generates the rebound response is aided by the presence of the transient K+ current. (4) A low-threshold, rebound, somatic Ca 2+ conductance [g Ca(T) ]. This conductance is inactive at the resting potential and deinactivates with hyperpolarization. (5) A high-threshold, dendritic Ca 2+ conductance [G Ca(P/Q) ].
This conductance triggers all-or-none depolarizing responses followed by activation of a Ca-gated K conductance. (6) A somatic h-type potassium channel.
In contrast to the IO, the high threshold Ca 2+ conductance is not strong in thalamic cells. Because of this difference, the dendritic Ca 2+ -dependent spike does not dominate the firing of the thalamic neuron, allowing it a wider range of firing properties that that in IO neurons. The amplitude of the dendritic Ca 2+ channels current in thalamic dendrites is smaller than that in inferior olivary neuron.
The six conductances combine to give thalamic neurons their unique oscillatory properties, as diagramed in Figure 12. At membrane potentials positive to −55 mV, fast action potentials are generated (red traces). At membrane levels near −55 mV, two types of firing are seen (black traces). In one case, the FIGURE 12 | Conducances underlying the oscillatory properties of thalamic neurons. In black, usual Na + dependent spike is followed by an after hyperpolarization generated by the classical voltage-sensitive K + conductance. Depending on membrane potential, this event can be followed by a persistent sodium current [(g Na(Ip) ) black spikes]. Following an inhibitory synaptic potential a hyperpolarization dependant deinactivation of type a g Ca(T) conductance, and the simultaneous inactivation of the potassium conductance g K(Ih) together generate a rebound response (green). The membrane potential is brought back to the threshold for the fast spike by the slow potassium conductance. In addition to the 10-Hz oscillations, slower oscillations (about 6 Hz) can occur by the rebound excitation (blue trace) following hyperpolarization of the cell [g K (Ca)] and inhibitory postsynaptic potentials (IPSPs). Such hyperpolarization deinactivates the low-threshold Ca 2+ conductance generating a rebound low threshold spike, which triggers the process once again activating potassium conductance (Ih). (Jahnsen and Llinás, 1984b,c;McCormick and Prince, 1986).
fast sodium-dependent spike is followed by an AHP, due to an increase in both the classical voltage-activated potassium conductance (g K ) and by a Ca 2+ -activated K + conductance [g K (Ca) ] that generates an AHP lasting for 70 ms or so, allowing the cell to fire at a frequency near 10 Hz. The response can be further augmented as a rebound from the inhibitory postsynaptic potential (IPSP) in blue.
Thalamic cell firing is basically produced by a slow depolarization of the cell produced by the activation of the persistent Na+ conductance [g Na(IP) ], which can serve as a continuous depolarizing drive once it is activated. Once the [g Na(IP) ] takes over it depolarizes the cell until another spike is generated and the process repeats itself, with a 10-Hz rhythmicity. If, on the other hand, the hyperpolarizing potassium conductances are combined with an A potassium current and/or IPSPs, the neurons are hyperpolarized sufficiently to deinactivate the low-threshold Ca 2+ conductance [g Na(Ip) ] and to inactivate a potassium conductance (Ih) resulting in an oscillatory responses at frequencies near 6 Hz. Thus, their intrinsic properties allow thalamic neurons to display a versatility whereby they switch between tonic and phasic responses as diagrammed in Figure 12.
The point to be emphasized here is not the difference between these two groups of cells but rather the fact that they both have intrinsic properties that give them distinctive firing characteristics. From the above, it follows that the nervous system  .
is constantly in action and that the patterns of activity arising from the sensory inputs and from the corollary discharge of motor outputs, are but a small modulatory component of the overall activity of the brain. Beyond these conductances, the thalamic neuron oscillatory patterns can also be generated via synaptic activation as elegantly demonstrated in vitro studies by Sohal et al. (2006).

CORTICAL NEURONS
The electrophysiology of cortical neurons has been extensively studied (Yuste et al., 2005) and the morphology-related intrinsic firing patterns in simulated neocortical pyramidal cells has been examined as well (Korogod and Tyc-Dumont, 2009). In this summary I will touch briefly on neuronal aspects of cortical neurons that relate very specifically to 40 Hz activation in relation to the intrinsic properties of a particular type of interneuron, the sparsely spinous neurons of the fourth cortical layer.
From an in vitro point of view, our research in the cerebral cortex of the guinea pig points to the existence of neurons in the fourth layer that have intrinsic subthreshold electroresponsive properties that endow these cells with a 30-to 45-Hz membrane potential oscillation . These cells, which are often silent after penetration, demonstrate oscillation on direct membrane depolarization. On occasion, the cells may also show spontaneous oscillations at that frequency. When this occurs, further depolarization produced by direct current injection will generate a spike at the peak of the depolarizing phase of each oscillation. In other recordings in similar neurons, it was also found that a voltage-dependent persistent sodium conductance may underlie the generation of 40-Hz oscillation, which, in that case, outlasts the duration of the depolarizing pulse.
Examples of such recordings are shown in Figure 13A. Autocorrelation analysis of this response ( Figure 13B) demonstrates that the frequency of oscillation of this cell was 42-Hz.
Following intracellular staining, these fourth-layer neurons were recognized as the sparsely spinous neurons that have been described by anatomists as being GABAergic and as having axons that ascend to the third layer and descend to the fifth layer in the cortex (Peters and Saint-Maie, 1984 As opposed to the oscillations observed in the IO and thalamus, the oscillation of these fourth-layer inhibitory interneurons appears to be generated by a voltage-dependent sodium conductance followed by a potassium conductance, probably of the voltage-or Na + -dependent variety. The sodium dependence of this oscillation was demonstrated by the addition of TTX to the bath, which blocked the oscillations generated by direct stimulation as well as those spontaneously generated. The subthreshold oscillations were, in fact, the first to disappear, followed by the blockage of the all-or-none fast spikes. Other examples of similar types of oscillations, but with a lower frequency, have been observed in the giant stellate cells of the entorhinal cortex (Alonso and Llinás, 1992) as well as in the neurons of the nucleus parabrachialis in the brainstem (Leonard and Llinás, 1990). In contrast to the 40-Hz oscillations displayed by the cortical neurons, the latter two cell types oscillate at a frequency of 6-12 Hz, i.e., in the range of the theta and low alpha rhythms. The point of interest here is that similar current may generate different oscillatory frequencies, depending on the kinetics of the voltage-dependent conductances. Equally significant is the fact that oscillations at a similar frequency may have different ionic bases in different types of cells. This is probably related to secondary events that oscillation may ultimately regulate. Thus, as some cell oscillations may be a form of communication, in others, the hippocampus for example, oscillation may serve to trigger secondary changes such as long-term potentiation (Larson and Lynch, 1986). These latter require the activation of Ca 2+ -dependent second messengers, in which case Ca 2+ -based oscillation would be a significant parameter (Llinás and Steriade, 2006).
In vivo cortical studies that have taken a broader perspective than that provided by single cell in vitro electrophysiology have demonstrated in the cat, that high-frequency activity occurs in motor areas 4y, 6aB and in the posterior parietal associative area 5a during motionless focused attention (Bouyer et al., 1987). These recordings were obtained from the surface of the cortex, as well as from depth field analyses. With respect to the visual cortex, it was demonstrated (Gray et al., 1989) that, following specific visual input, a 40-Hz oscillation may be observed as a field potential envelope, and as single units in the overall envelope. This 40-Hz oscillation appears to be present only when the given cortical area is activated by an optimal physiological stimulus.
The question of the role of the intrinsic electrical properties of neurons in the overall function of the CNS must be defined, then, at the cell ensemble level. Perhaps one of the most interesting issues concerning global brain function relates to the rediscovery in recent years of 40-Hz oscillation, which may be observed in the cortex under certain conditions. In fact, 40-Hz oscillations have been observed in the cortex under certain conditions. For example, there are 40-Hz oscillations during physiological stimulation of the visual (Gray et al., 1989) or auditory cortex (Galambos et al., 1981;Spydell et al., 1985;Mäkelä and Hari, 1987;Johnson et al., 1988). In its absence, as occurs when P type calcium channels are (Ca 3.1) are deleted, there is a total lack of cognitive function. Such oscillations have also been recorded during intensive attention states such as occur when a predator is stalking its prey (Bouyer et al., 1987), or in humans, during the state of elevated attention prior to the execution of complicated tasks (Sheer, 1984). Similar activity as measured from the scalp by electric and magnetic means in humans appears to be well correlated with cognitive tasks and seems to be altered under pathological conditions (Ribary et al., 1989(Ribary et al., , 1991.
The issue here is that the oscillatory events may be used in intercellular communication as well as in the modulation of the intracellular milieu. From the point of view of 40-Hz oscillations, the oscillation in the GABAergic neurons may, in fact, be the origin of the macroscopic 40-Hz observed in the cortex. Indeed, since these cells receive direct synaptic input from thalamic neurons, and since they relay inhibition to pyramidal cells at a 40-Hz frequency, this will, in turn, produce a resonance activation of the pyramidal cells at a frequency of 40 Hz. The question of interest, then, is the mechanism by which such oscillations may become sufficiently synchronous to generate macroscopic events. Our own view on this matter is illustrated in Figure 14.
Thalamic input via projection neurons reaches the neurons of the fourth layer of the cortex that generate 40-Hz oscillation. These cells, which are GABAergic, can generate a 40-Hz IPSP on pyramidal neurons and allow them to fire at 40-Hz as a rebound from abrupt inhibition. Pyramidal cells, in turn, activate, via collaterals, the thalamic projection neurons as well as the neurons of the reticular thalamus and the interneurons at the thalamus itself. The direct excitatory input at 40-Hz to the projection thalamic cells, as well as their disynaptic inhibition via the reticularis and intrathalamic GABAergic neurons, contributes to the thalamic oscillations at 40-Hz. This thalamic oscillation is then signaled back to the cortex, establishing a large resonant oscillation between the thalamus and the cortex, which can recruit sufficient elements to generate the synchronicity observed at both intracellular and extracellular levels in the cortex and thalamus. According to this view, the inhibitory neurons that contact pyramidal cells directly may force them in combination to a synchronous excitatory input to rebound oscillation. These cells will then generate, via their descending axons, 40-Hz excitation of cells in the nucleus reticularis thalami (NRT), the intrinsic inhibitory neurons in the thalamus, and the projection thalamic neurons themselves.
Since the NRT cells are inhibitory (cf. Steriade and Llinás, 1988) they would further increase the resonance property of the thalamocortical system by their own driven 40-Hz input back through the fourth-layer interneurons. In this manner, a corticothalamo-cortical resonance even may actually be the basis of this 40-Hz rhythm recorded at the cortical level.
While Gray et al. (1989) have not recorded oscillation in the thalamus at 40-Hz, other investigators (Fuster et al., 1965) had, in fact, published intracellular recordings from geniculate neurons following light stimulation demonstrating clear 40-Hz activity. This indicates that activity at such frequencies is not a cortical phenomenon exclusively but may also be seen at the thalamic level.
The MEG recordings shown in Figure 15 suggest that mostly somatosensory and visual auditory association cortices are active as well as the anterior temporal pole (amygdala) are the main players during dreaming while the frontal lobe remains only sparsely active (Figure 15F). Similar findings have been reported using MEG by Ioannides et al. (2004).
The functional significant of 40-Hz oscillation in the thalamocortical system becomes particularly interesting when one considers that it may serve as the basis for the temporal correlation of events that must be considered as a single perceptual or motor entity, the so called conjunction principle. This temporal superposition, in fact, may be at the very core of global brain function. Indeed, it may actually be observed using magnetic recording in humans, where 40-Hz activity can be demonstrated to be organized quite widely, demonstrating a phase shift from the front of the brain onto the back with a sweep speed in the rostro-caudal direction of about 5-10 ms (Ribary et al., 1989(Ribary et al., , 1991. This phase shift suggests that, via the activation of the NRT, a conjunctional type of activity may be generated at the thalamus, which allows thalamocortical resonance in a global, organized manner. This, in its most simplified form, Frontiers in Cellular Neuroscience www.frontiersin.org November 2014 | Volume 8 | Article 320 | 12 could serve to scan the brain front to back 200 times a second. Such scanning would be viewed as the basis for the generation of unitary perceptual entities out of many sensory and motor vector components, which represent the details of the perceived world. Ultimately, we are then faced with a system that addresses the external world, not as a slumbering machine to be awoken by the entry of sensory information, but rather as a continuously humming brain willing to internalize and incorporate into its intimate activity an image of the external world, but always in the context of its own existence and its own intrinsic electrical activity. Most fundamental, however, is the fact that the system is not merely a computational entity, but rather that issues are substrate dependent (neurons) and, moreover, that ionic events such as the presence of P/Q calcium channel activity are actually crucial to its ultimate function. From a more global perspective it is the dialog between the incoming information arising, in mammals, from the dorsal thalamus that provides the content in our everyday cognitive activity, and the nonspecific system that provides context (i.e., the attention), that we give to such inputs (Llinás et al., 1994).