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Over the last few decades intensive studies have been carried out on the molecular targets mediating general anesthesia as well as the effects of general anesthetics. The γ-aminobutyric acid type A receptor (GABAAR) has been indicated as the primary target of general anaesthetics such as propofol, etomidate and isoflurane, and sedating drugs including benzodiazepines and barbiturates. The GABAAR is also involved in drug tolerance and dependence. However, the involvement of other ion channels is possible.
Using reverse transcription and quantitative PCR techniques, we systematically investigated changes in the mRNA levels of ion channel genes in response to exposure to midazolam, pentobarbital and ketamine in a freshwater model animal, Daphnia pulex. To retrieve the sequences of Daphnia ion channel genes, Blast searches were performed based on known human or Drosophila ion channel genes. Retrieved sequences were clustered with the maximum-likelihood method. To quantify changes in gene expression after the drug treatments for 4 hours, total RNA was extracted and reverse transcribed into cDNA and then amplified using quantitative PCR.
A total of 108 ion channel transcripts were examined, and 19, 11 and 11 of them are affected by midazolam (100 μM), pentobarbital (200 μM) and ketamine (100 μM), respectively, covering a wide variety of ion channel types. There is some degree of overlap with midazolam- and pentobarbital-induced changes in the mRNA expression profiles, but ketamine causes distinct changes in gene expression pattern.
In addition, flumazenil (10 μM) eliminates the effect of midazolam on the mRNA expression of the GABAA receptor subunit Rdl, suggesting a direct interaction between midazolam and GABAA receptors.
Recent research using high throughput technology suggests that changes in mRNA expression correlate with delayed protein expression. Therefore, the mRNA profile changes in our study may reflect the molecular targets not only in drug actions, but also in chronic drug addiction. Our data also suggest the possibility that hypnotic/anesthetic drugs are capable of altering the functions of the nervous system, as well as those non-nerve tissues with abundant ion channel expressions.
Keywords:Midazolam; Pentobarbital; Ketamine; mRNA; Ion channel; Daphnia pulex
Midazolam is a benzodiazepine and widely used as an anxiolytic, anticonvulsant, sleep aid, muscle relaxant, and antipsychotic. Pentobarbital is a short-acting barbiturate that is used as a sedative and anesthetic agent. Like other barbiturates, pentobarbital produces a wide spectrum of dose-dependent effects, including sedation, hypnosis, anesthesia and finally coma. Although their use has decreased over the years because of high abuse potential, barbiturates are still being prescribed to many patients, such as epilepsy patients and people with sleep disorders. The principal mechanism of actions of benzodiazepines and barbiturates is believed to be positive allosteric modulation of the γ-aminobutyric acid (GABA) type A receptor (GABAAR) [1-3]. Ketamine, a rapid acting anesthetic agent and a popular drug of abuse, has diverse effects, including antidepressant action and analgesic effects on chronic pain [4]. These actions were traditionally believed to arise from the inhibition of NMDA receptors. However, NMDAR blockers, such as MK-801, fail to mimic the actions of ketamine [5,6]. Furthermore, knockout of NR2A subunit reduces but does not eliminate the actions of ketamine [7-9]. Ketamine has also been shown to positively modulate the function of the cerebellar GABAA receptors containing α6 and δ subunits [10].
Chronic use of midazolam, phenobarbital and ketamine produces tolerance and physical dependence. The homeostatic theory of drug tolerance [11] claims in a modern form that the functional tolerance results from altered function or expression of proteins in a way to reduce the effects of the drugs. Based on this theory, for example, the GABAAR, which is positively modulated by drugs such as benzodiazepines and barbiturates, is expected to be downregulated after prolonged or repetitive drug exposure. This adaptive response may involve, although not necessarily, reduction in the mRNA levels of the GABAAR subunits. Therefore, comparison of changes in mRNA expression patterns in response to different drugs may offer clues to molecular targets involved in drug actions and drug tolerance/dependence.
The effects of benzodiazepines and barbiturates on the mRNA expression of different GABAAR subunits have been investigated over the last few decades [12-17]. Studies of the transcriptional responses of the NMDAR genes to diazepam [18-20] and flurazepam [21] are also available. Unfortunately, such studies have led to conflicting results. For example, diazepam was found to downregulate GABAAR α1 mRNA expression in the cerebral cortex by several research groups [12-14], but others [17,22] found diazepam ineffective. Similar situations were found for other GABAAR subunits [23]. Diazepam was found to increase the cortical mRNA contents of NR1 and NR2B subunits [18-20]. However, another study showed decreases in hippocampal NR2B mRNA and protein after chronic flurazepam treatment [21]. Compared with the GABAAR and NMDAR, the effects of barbiturates, benzodiazepines and ketamine on the mRNA expression of other types of ion channels are largely unknown.
Ion channels are complex proteins forming ion-per-meable pathways through biological membranes. The ion channels tested in this study include the P-domain channels [24], the pentameric ligand-gated ion channels (pLGICs), the ENaC/Deg ion channels, the ATP-gated ion channels (P2X receptors), the calcium release-activated calcium (CRAC) channels, the inositol 1, 4, 5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), and the chloride ion channels. The only known ion channel genes not included in this study were the invertebrate ionotropic receptors (IRs), a variant subfamily of iGluRs [25,26].
The P-domain channels have a common pore architecture composed of four homologous pore-domains contributed by one, two or four subunits arranged in four-fold symmetry. This group of channels include voltage-gated potassium (Kv) channels, cyclic nucleotide-gated (CNG) channels, hyperpolarization-activated CNG (HCN) channels, voltage-gated calcium (CaV) channels, voltage-gated sodium (NaV) channels, sodium-leak channels (NALCN), two-pore channels (TPCs), transient receptor potential (TRP) channels, and glutamate-gated ion channels (or ionotropic glutamate receptors, iGluRs).
Most pLGICs are gated by extracellular ligands, and include the nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine type 3 receptors (5-HT3Rs) GABAARs and glycine receptors (GlyRs). The mammalian pLGIC superfamily also includes zinc-activated ion channels (ZACNs), the invertebrate pLGIC superfamily also includes the glutamate-gated chloride (GluCl) channels, histamine-gated chloride (HisCl) channels, and pH-sensitive chloride (pHCl) channels.
Both ENaC/Deg channels and P2X receptors are trimeric and share similar transmembrane topology. However, they are not homologous in amino acid sequences. The CRAC channels are hexameric plasma membrane proteins mediating the entry of extracellular Ca2+ when the intracellular Ca2+ stores are depleted. The IP3Rs and RyRs, on the other hand, are intracellular membrane proteins important to intracellular Ca2+ signaling. The chloride channels are anion permeable protein complexes (excluding the GABAAR and GlyR) and are less-well understood.
The aim of our study is to measure the effects of midazolam, pentobarbital and ketamine on the ion channel mRNA expression in the water flea Daphnia pulex, a freshwater crustacean with great potential for biomedical research (http://www.nih.gov/science/models/ webcite).
Daphnia cultures and treatments
The method for culturing D. pulex was the same as previously described [27]. Briefly, daphnids from a single clone were kept in a temperature- and photocycle-controlled tank (20 ± 1°C, 16:8 h light-dark cycle) and fed daily with a mixture of Saccharomycetes and Spirulina. One-fifth of the medium was renewed every second day. Midazolam, pentobarbital sodium and ketamine stock solutions of 10, 100 and 100 mM, respectively, were made in distilled water, and diluted to final concentrations before use. Since the constant jumping movements of Daphnia may complicate experimental results if the animals are immobilized by drugs while the animals in the control group are free to swim, drugs of subanesthetic concentrations based on the EC50 values (Figure 1) were used to investigate the transcriptional responses of ion channel genes. For this purpose, daphnids were exposed to midazolam (100 μM), pentobarbital (200 μM), and ketamine (100 μM) for 4 hours, and killed by crushing immediately for mRNA quantification by RT-qPCR. Protocols were approved by the Scientific and ethics committee of Sichuan university and adhered to the international guidelines for animal care.
Figure 1. Daphnia immobility dose responses of midazolam and midazolam + flumazenil (Panel A), pentobarbital and ketamine (Panel B). 20 daphnids were treated with midazolam, midazolam + flumazenil, pentobarbital and ketamine at series of concentrations. The numbers of immobilized daphnids were observed 4 hours after each treatment. The immobile ratio were presented as means ± SE (n = 3).
Sequence analysis and phylogenetic inference
The Daphnia genome (http://wfleabase.org/ webcite), NCBI and Uniprot protein database were searched for the Daphnia ion channel gene models based on known human or Drosophila genes. Sequences were analyzed with the maximum-likelihood (ML) method using MEGA 5.10 program [28]. The sequences used are designated in succession by the abbreviation of the species (Hs for Homo sapiens, Dm for Drosophila melanogaster, Ce for C. elegans, Am for Apis mellifera, and Dpul for Daphnia pulex) and the gene name.
RNA extraction, reverse transcription and polymerase chain reaction (PCR)
Total RNA extraction, reverse transcription and PCR were performed as previously described [27]. Briefly, RNA was harvested from 50 crushed daphnids for phase separation, precipitation, and quantification. cDNA was generated using PrimeScriptTM RT reagent Kit DRR037A (TaKaRa), and amplified first by regular PCR to screen primers, which were designed using Primer 3 software [29] based on the scaffold sequences (http://wfleabase.org/ webcite). Successful primers (Additional file 1: Table S1) were then used for qPCR to quantify changes in gene expression after drug treatment. qPCR was carried out on an iQ5 system (Bio-Rad) using SYBR Premix Ex TaqTM II KIT DRR081A (TaKaRa). Each reaction was run in triplicate and contained 2 μl of cDNA template along with 0.8 μM primers in a total volume of 20 μl. Cycling parameters were 95°C for 30 s to active the DNA polymerase, then 40 cycles of 95°C for 5 s, 55°C for 30 s and 72°C for 30 s. Melting-curves were performed to verify only a single product without primer-dimers. Data were normalized against a house-keeping reference gene β-actin, and were analyzed using the 2-ΔΔCT method [30].
Additional file 1: Table S1. Gene name, Gene ID, Genome Map, Primer, product size and qPCR efficiency. The forward and reverse primers, gene ID, product size and qPCR efficiency for indicated genes were given, based on the scaffold sequences of the Daphnia Genomics Consortium. All primers were designed using Primer 3 (version 0.4.0). The expression and length of each gene were confirmed by reverse transcription PCR. qPCR efficiency of target genes was calculated by Bio-Rad iQ5 software.
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Statistical analysis
All data were presented as means ± SE. qPCR data represent the average of 5 replicate experiments; all results were normalized to β-actin, an internal control, and then to control group. Differences in relative expression of genes were assessed using paired-t test (n = 5). Statistical significance was set at a level of P < 0.05 (*) and P < 0.01 (**).
Phylogenetic analysis
More than 120 gene models of ion channel proteins have been predicted in the Daphnia genome and 108 of them were successfully amplified by RT-PCR (Additional file 2: Table S2). For comparison purposes, these proteins are classified into different categories based on sequence homology molecular structure, and ion selectivity in the case of chloride channels.
Additional file 2: Table S2. Gene name, synonyms of the gene name and Uniprot accession numbers used in phylogenies.
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The four-fold symmetric P-domain ion channels
A total of 53 P-domain channel genes are detected at transcript level in Daphnia (Additional file 2: Table S2), and they are classified into four superfamilies: Kv/CNG, CaV/NaV, TRP and iGluR. The Kv/CGN members are further classified into three families based on subunit transmembrane topology: the two transmembrane-helix (2TM) family (Additional file 3: Figure S1), the 6TM family (Additional file 4: Figure S2), and the 4TM (K2P) family (Additional file 5: Figure S3). The 4TM potassium genes resulted from duplication of 2TM genes during the evolution, thus the name K2P, while the 6TM domain consists of a 2TM domain and a voltage sensor domain. The CNG and HCN channels are homologous to the 6TM Kv channels (Additional file 4: Figure S2). The CaV or NaV channel contains a single principal subunit with four 6TM domains. The NALCN and TPC channels also belong to the NaV/CaV superfamily. The NALCN is a voltage-independent, TTX-insensitive, and nonselective cation channel underlining the background Na+ leak current [31]. TPC channels are intracellular ion channels mediating the second messenger NAADP-regulated Ca2+ release. The Daphnia NaV/CaV superfamily contains 7 members: 3 CaV, 2 NaV, 1 NALCN and 1 TPC genes (Additional file 6: Figure S4).
Additional file 3: Figure S1. Phylogenetic tree of the 2TM-KV (Kir) channels. The Kir subunit possesses a P-domain composed of two transmembrane helices connected by a P-loop. Four Kir subunits assemble to for a channel. The mammalian Kir family comprises the classical inward-rectifying K+ channels (Kir2), the KATP channels (Kir6), G protein-activated K+ channels (Kir3), and K+-transport channels. The invertebrate Kir genes form distinct clusters. The Daphnia genome contains two Kir genes: Dpul_Ir and Dpul_Irk2. The homolog of the Drosophila Irk3 is absent in Daphnia genome. Abbreviation: Hs, Homo sapiens; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Dpul, Daphnia pulex.
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Additional file 4: Figure S2. Phylogenetic tree of the 6TM-Kv, CNG and HCN channels. The 6TM-Kv channels are highly diverse in sequence, structure and function. In addition to the P-domain, each 6TM-KV subunit obtains a voltage sensor domain (VSD) composed of four transmembrane helices. The CNG and HCN channels, although not classified as Kv channels, are homologous to the KCNH KV channels. Daphnia and Drosophila share similar gene sets for the 6TM-KV/CNG/HCN channel group, with subtle differences. The homolog of the Drosophila elk is absent in the Daphnia genome. Daphnia have two homologs of the Drosophila Shawl genes, namely Shawl1 and Shawl2, while Drosophila have one.
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Additional file 5: Figure S3. Phylogenetic tree of the 4TM-KV (K2P) channels. The K2P subunit consists of two P-domains, and two such subunits assemble to form a channel. Daphnia have five K2P members: Dpul_TWIK, Dpul_Task6, Dpul_Task7, Dpul_Ork1 and Dpul_TRESK. The Daphnia homolog of the mammalian TWIK is detected at transcript level, but the Drosophila counterpart is absent. As in Drosophila, the TREK member of the K2P channels is also absent in Daphnia. Abbreviation: Hs, Homo sapiens; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Dpul, Daphnia pulex.
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Additional file 6: Figure S4. Phylogenetic tree of the CaV, NaV, NALCN and TPC channels. The CaV and NaV channels are thought to arise from the potassium channels during evolution. The CaV/NaV channel contains a single principal (α1) subunit with four 6TM domains. The human genome contains ten α1 genes, which can be further clustered in three groups: CaV1 (L-type), CaV2 (P/Q, N, and R type), and CaV3 (T-type). The Daphnia genome predicts three genes: Dpul_Ca-alpha1D (CaV1), Dpul_Ca-alpha1T (CaV2) and Dpul_cac (CaV3). NALCN is represented by a single gene in human, Daphnia and Drosophila. Unlike the CaV, NaV and NALCN, the TPC subunit contains two P-domains and assembles as a dimer. The transcript of the TPC encoding gene is detected in Daphnia. TPC Gene models are also available for many insects, such as Apis mellifera, but absent in Drosophila. Abbreviation: Hs, Homo sapiens; Dm, Drosophila melanogaster; Am, Apis mellifera; Caenorhabditis elegans; Dpul, Daphnia pulex.
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TRP channels are highly diverse in function, structure and distribution, with 28 mammalian TRP genes classified into six subfamilies: TRPC, TRPV, TRPM, TRPML, TRPA and TRPP. In addition, the invertebrates have a group known as TRPN, which is also found in zebrafish. The Daphnia genome contains 13 TRP subunit genes (Additional file 7: Figure S5).
Additional file 7: Figure S5. Phylogenetic tree of the TRP channels. Despite of the structure and function diversities, all TRP subunits have a common design for the transmembrane domain, which basically is a 6TM domain. So far 28 mammalian TRP subunit encoding genes are known and belong to six subfamilies known as the TRPC, TRPV, TRPM, TRPML, TRPP and the TRPA, and all these groups have invertebrate orthologs. The Daphnia genome predicts 13 TRP subunit genes and all were detected at transcript level, including a 3 TRPC-like genes (Dpul_TRP, Dpul_TRPL and Dpul_Trpgamma), 2 TRPM-like gene (Dpul_TRPM1, Dpul_TRPM2), 4 TRPA-like genes (Dpul_TRPA5, Dpul_pain, Dpul_pyx1, Dpul_pyx2), 3TRPV genes (Dpul_nan, Dpul_iav, TRPV_nompc), 1TRPML gene (TRPML). TRPP genes are absent in Daphnia. Abbreviation: Hs, Homo sapiens; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Am, Apis mellifera; Dr, Danio rerio; Dpul, Daphnia pulex.
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The iGluRs are tetrameric and can be divided into three subfamilies based on pharmacology and homology: AMPA, NMDA and kainate (KA) receptors. The Daphnia genome contains eight iGluR homologs: Dpul_Glu-RI, Dpul_Nmdar1-3, Dpul_KaiR1-4 (Additional file 8: Figure S6). A variant iGluR subfamily, the ionotropic receptors (IRs), was not included in this study.
Additional file 8: Figure S6. Phylogenetic tree of the iGluRs. The iGluR is a heterotetramer containing four P-domains, each in one subunit. The mammalian iGluRs are divided into three groups: AMPA, NMDA and KA receptors. There are two other classes with respect to sequence similarity known as the delta class (GRID) and the kainate-binding proteins (KBP). The invertebrate iGluRs display considerable variations, especially in the KAR branch. In addition, a GRIN3-like gene is detected in Daphnia at the transcript level, but it is absent in Drosophila. Abbreviation: Hs, Homo sapiens; Dm, Drosophila melanogaster; Xl, Xenopus laevis; Gg, Gallus gallus; Dpul, Daphnia pulex.
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The pentameric ligand-gated ion channels
The Daphnia genome contains 20 pLGIC genes: 12 nAChR (Additional file 9: Figure S7), 5 GABAAR (Grd, Rdl, RdlL, Lcch3 and CG8916), 1 GluCl, and 2 HisCl genes (Additional file 10: Figure S8). There is no RdlL counterpart in Drosophila. The classification of the Daphnia pLGICs into nAChR and GABAAR groups is simply based on homology.
Additional file 9: Figure S7. Phylogenetic tree of the nAChRs. The nAChRs belong to the Cys-loop receptor superfamily. The nAChRs are highly diverse in both vertebrates and invertebrates. Daphnia genome encodes 12 putative nAChR genes. Abbreviation: Hs, Homo sapiens; Dm, Drosophila melanogaster; Gg, Gallus gallus; Am, Apis mellifera; Dpul, Daphnia pulex.
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Additional file 10: Figure S8. Phylogenetic tree of non-nAChR cys-loop receptors. In addition to the nAChRs, the Cys-loop receptor superfamily also includes 5-hydroxytryptamine type 3 receptors (5-HT3Rs), zinc-activated ion channels (ZACNs), GABAARs and glycine receptors (GlyRs). The Daphnia genome lacks the counterparts of the Drosophila genes CG11340, CG6927 and CG7589, which form a separate branch. The pHCl gene seen in Drosophila is also absent in Daphnia.
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The ENaC/Deg channels and P2X receptors
The ENaC/Deg channels and P2X receptors are distinct classes of trimeric protein complexes. The Daphnia ENaC/Deg homologs are quite diverse, with 14 members detected at the transcript level (Additional file 11: Figure S9). Two Daphnia P2X genes are detected at the transcript level (Dpul_P2XL1 and Dpul_P2XL2 (Additional file 12: Figure S10).
Additional file 11: Figure S9. Phylogenetic tree of the Deg/ENaC channels. DEG/ENaC channels have been found in nematodes, insects and vertebrates and implicated in a broad spectrum of cellular functions. Mammalian DEG/ENaC channels fall into two major groups (EnaC and ASIC) with 9 members. The invertebrate DEG/ENaC members are highly diverse. Fourteen Daphnia ENaC/Deg homologs are detected at transcript level.
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Additional file 12: Figure S10. Phylogenetic trees of the P2X receptor, ORAI proteins, IP3Rs and RyRs. Two P2X gene transcripts are detected in Daphnia, meanwhile, one ORAI, one IP3R and one RyR are detected in Daphnia.
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The CRAC channels, IP3Rs, RyRs and the chloride channels
The Daphnia genome contains one CRAC channel gene, one IP3R gene, and one RyR gene (Additional file 12: Figure S10). There are at least five distinct classes of Cl– channels, including the ClC channels, the CLIC proteins, bestrophin, the tweety chloride channels, and anoctamin/TMEM16. The Daphnia genome contains 7 ClC genes, 2 CLIC genes, 4 bestrophin genes, 1 tty gene and 3 TMEM16 genes (Additional file 13: Figure S11).
Additional file 13: Figure S11. Phylogenetic trees of the CLC, CLIC, BEST, tweety and ANO proteins. Chloride channels are a functionally and structurally diverse group of anion selective channels. Chloride channels are poorly understood and classified into ClC, Clic, Best, Tweety and Ano families. There is no sequence homology between these families.
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Dose-dependent immobility upon midazolam, pentobarbital, and ketamine treatment
In order to determine the subanesthetic concentrations for chronic treatment in our mRNA assay, daphnids were exposed to aquarium water containing a series of concentrations of midazolam, pentobarbital, or ketamine for four hours (Figure 1). Midazolam, pentobarbital and ketamine immobilized the daphnids at EC50 values of 0.65, 0.92 and 0.84 mM, respectively. Flumazenil (10 μM), a competitive antagonist of benzodiazepines and used to reverse the actions of benzodiazepines in clinical settings, shifted the dose-response curve of midazolam to the right (EC50 = 0.86 mM , Figure 1A), but had no effects of its own on the immobility of the daphnids up to 100 μM. Interestingly, ketamine at concentrations of >200 μM produced a consistent circling behavioral phenotype, mimicking the core behavior aspects of rodents and fish administered with ketamine [32,33]. This aberrant behavior was completely absent in the control daphnids. Ketamine-induced immobility started at higher concentration (~400 μM). Based on these dose-dependent responses, we exposed daphnias to 100-μM midazolam, 200-μM pentobarbital and 100-μM ketamine for 4 hours for our mRNA assays. At these concentrations, even longer treatment (10 hours) did not result in death of the animals. The use of subanesthetic concentrations instead of anesthetic ones were due to the fact that daphnids jump constantly and this behavior may complicate the experimental results if the animals were immobilized by drugs while the animals in the drug-free (control) group were free to swim.
Effects of midazolam on the transcription of daphnia ion channel genes
Out of the 108 genes tested, the transcription of 6 genes was upregulated by midazolam treatment, including the 6TM Kv channel shal, the CNG channel cngl, the nAChR subunit ACHA4, the GABAAR RdlL, DEG/ENaC-11 and P2XL1 (Figure 2). Meanwhile, midazolam downregulated transcription of 13 genes, including the 6TM Kv channels shaker and shawl1, the CNG channel CNGA1, the TRP channels nan and iav, the NMDA receptor Nmdar1, the GABAAR Rdl and GRD, the HisCl hclB, the CLC Clc-c1, Clc-c2, DEG/ENaC-10 and IP3R (Figure 2A). Flumazenil (10 μM) abolished the effects of midazolam on the mRNA expression of Rdl, Shawl1 and Shaker (Figure 2B). Flumazenil alone, however, did not affect the Rdl, Shawl1 and Shaker mRNA expression. In addition, we found that flumazenil (10 μM) alone downregulated Nmdar1 and IP3R transcription (Figure 2C).
Figure 2. Effects of midazolam and flumazenil on the expression of Daphnia ion channel genes. (A). Daphnids were exposed to aquarium water containing 100 μM midazolam for 4 hours and killed for mRNA quantification. Out of 108 ion channel genes, 19 genes are affected by midazolam. (B). Daphnids were co-treated with flumazenil (10 μM) along with midazolam (100 μM) for 4 hours and killed for mRNA quantification. Flumazenil abolishes the effects of midazolam on the mRNA expression of Rdl, Shawl1 and Shaker. (C) Daphnids were exposed to 10 μM flumazenil alone for 4 hours before mRNA quantification. The Nmdar1 and IP3R transcripts were significantly reduced by flumazenil. Statistical significance was set at a level of P < 0.05 (*) and P < 0.01 (**).
Effects of pentobarbital on the transcription of daphnia ion channel genes
Pentobarbital (200 μM) downregulated the expression of the 6TM Kv channel genes Shawl1 and Shawl2, the CaV channel gene Ca-alpha1D, the NMDAR Nmdar1, the GABAARs Rdl and GRD, the CLC Clc-c2, while the KAR gene KaiR1 and KaiR4, the AMPAR gene Glu-RI and the nAChR ACHA4 were upregulated by pentobarbital (Figure 3).
Figure 3. Effects of pentobarbital on the expression of the Daphnia ion channel genes. Daphnids were exposed to 200 μM pentobarbital for 4 hours and killed for mRNA quantification. Seven genes are downregulated and four genes are upregulated at transcriptional level by pentobarbital. Statistical significance was set at a level of P < 0.05 (*) and P < 0.01 (**).
Effects of ketamine on the transcription of daphnia ion channel genes
Ketamine (100 μM) downregulated transcription of 11 genes, including the 2TM Kir channels Ir and Irk2, the K2P channels Task6, Ork1 and TRESK, the TRP channel Trpgamma, the iGluRs Glu-RI and KaiR1, the CLC CLC-c1 and CLC-c2, and tty (Figure 4).
Figure 4. Effects of ketamine on the expression of Daphnia ion channel genes. Daphnids were exposed to 100 μM ketamine for 4 hours and killed for mRNA quantification. Among 108 ion channel genes, ketamine affects 11 genes at transcriptional level. Surprisingly, all of the 11 genes are downregulated by ketamine. Statistical significance was set at a level of P < 0.05 (*) and P < 0.01 (**).
Invertebrate model organisms, such as C. elegans[34] and Drosophila[35], are desirable models for the studies of the mechanisms of drug tolerance/dependence. These simple animals share many complex traits with mammals, even at behavioral levels [36]. Furthermore, the major groups of ion channels are conserved, simplifying the dissection of core molecular machinery responsible for drug addiction.
The choice of midazolam concentration (100 μM) for the mRNA assays was based on the dose-response curve (Figure 1). Compared to clinic concentrations, which are around 1 μM [37], the high midazolam concentration may result in nonspecific effects of the drug and consequently in changes in gene expression that are not related to midazolam action on GABAA receptors. Nonetheless, the down-regulation of the Rdl mRNA by midazolam appears to be directly mediated by GABAA receptors, since this effect is eliminated by flumazenil (10 μM), a competitive antagonist of benzodiazepines. In addition, the direction of Rdl mRNA regulation is consistent with the prediction by the homeostasis theory of drug tolerance [11]. Despite the role of mRNA regulation is not explicitly implied in the theory, recent studies suggest that protein and mRNA expression levels are correlated [38-40]. Interestingly, flumazenil also eliminates midazolam-induced reduction in the mRNA levels of Shawl1 and Shaker (Figure 2B), suggesting a possibility of crosstalk between GABAAR and potassium channel signalings.
The short time course of drug treatment in the current study may undermine the relevance of the results to questions of addiction and drug dependence. For the development of tolerance and dependence in mammals, chronic treatments lasting weeks to months are required. In Drosophila, the lifespan of which is around 30 days under common culture conditions, the time courses of chronic treatments are much shorter, but vary greatly depending on experiment scenarios and the purposes of the study. For example, induction of neurodegeneration mimicking Parkinson's disease requires days of treatments [41,42]. Alcohol addiction in flies, on the other hand, develops more rapidly. Two types of addiction have been identified [35]: rapid tolerance can be induced by a single brief (less than 60 min) exposure to ethanol, while chronic tolerance requires prolonged (~24 h) ones. Chronic alcohol tolerance depends on protein synthesis, while molecular events downstream of protein synthesis contribute to acute tolerance. However, in general the emerging picture of the regulation of mRNA and protein expression is perhaps more complex than initially thought [43]. Accumulating evidence shows that mRNA expression correlate best with delayed protein expression. For example, the mRNA abundance changes in yeasts occur in a time window of 20 to 240 min after rapamycin treatment, while the protein abundance changes mostly occur at 4 and 6 h of the treatment [44]. This temporal correlation between mRNA and protein expression is not specific to rapamycin, since oxidative stress also induces rapid transcriptional changes which peak at 60 min after the treatment and decay quickly, while the protein expression response is much slower [38]. Nothing is known about the temporal relationship between mRNA and protein abundances in the development of chronic drug tolerance. However, it is highly possible in our case that changes in ion channel mRNA levels may lead to delayed alteration in protein abundance and chronic response to the drug treatments.
Our results suggest that Daphnia are feasible model animals for the investigation of the role of GABAA receptors in drug addiction, including addiction to benzodiazepines. Many early studies suggest that insect GABAA receptors were relatively insensitive to benzodiazepines. These studies involved the use of recombinantly expressed GABAA receptors, such as the homo-oligomeric Rdl receptor [45,46] and the Grd/LCCH3 receptor [47]. There is, however, evidence that native insect GABAA receptors are modulated by some benzodiazepines. For example, Lees et al. (1987) [48] showed that the 7-nitro benzodiazepine flunitrazepam enhanced the amplitude of GABA-induced currents by up to 70%. More recently, Buckingham et al. (2009) [49] observed that GABA-induced currents in acutely dissociated insect motor neurons were enhanced by both Ro5-4864 and diazepam, whereas clonazepam was ineffective. The pharmacological differences between recombinant and native GABAA receptors suggest more complex subunit combinations in native GABAA receptors. The involvement of GABAA receptors in benzodiazepine dependence appears to be evolutionarily conserved. It is observed in flatworms, the simplest bilaterian animals, that flumazenil (10 μM) antagonizes the abstinence-induced withdrawal from midazolam (10 μM), but has no effect of its own on the behavior of the animals [50].
In Daphnia, ketamine at lower concentrations produces increased movements in the form of circling swimming, while immobility starts at higher concentrations. The dissociation of hyperactivity from immobility is interesting and raises the possibility that the molecular targets responsible for ketamine-induced hyperactivity are different from those for immobility. Currently, we found that ketamine-induced changes in the mRNA expression profile are quite different from those seen with midazolam and pentobarbital treatments. It is worth noting that most of the Daphnia genes affected by ketamine are from the P-domain channel group, including the 2TM (Ir, Irk2), 4TM (Task6, ork1, TRESK), TRP (Trp-gamma), iGluR (Glu-R1, KaiR1) families. A P-domain is basically the 2TM domain and composed of two transmembrane helices connected by a loop region referred to as the P-loop. Four P-loops in a ion channel form the pore selectivity filter. It is believed that the open-channel blockers of NMDARs, such as MK-801 and ketamine, bind to the P-loop amino acid residues. It is therefore highly possible that ketamine may interact with the other P-domain channels in a similar way. The K2P channel, ork1, has been implicated in the regulation of cardiac automatic activity in Drosophila[51], as well as in mammals [52]. In mammals, the kainate receptors are implicated in chronic pain regulation [53]. The Daphnia KaiR1 gene is homologous to the mammalian KARs and was downregulated at the transcript level. Unfortunately, data regarding the physiology of KaiR1 is unavailable.
Compared with the P-domain channels and pLGICs, other ion channels are less well understood. Nonetheless, recent studies suggest that the CLC chloride channels play a role in synaptic transmission and plasticity [54,55], as well as in neuronal excitability [56]. We found in Daphnia that the Clc-c2 mRNA expression was downregulated by midazolam, pentobarbital and ketamine, while the CLC-c1 mRNA expression was also downregulated by midazolam and ketamine, suggesting a new type of molecular targets possibly involved in drug addiction.
The major groups of ion channels are highly conserved across the animal kingdom. In addition, simple animals, such as C. elegans and Drosophila, share many complex traits with mammals, even at behavioral levels [36]. Therefore, the use of invertebrate model organisms may greatly simplify the dissection of core molecular machinery responsible for drug actions and addiction. In Daphnia, midazolam, pentobarbital and ketamine cause distinct mRNA expression profiles and this observation provide insights into potential novel molecular targets involved in drug actions and addiction.
Competing interests
The authors declare that they have no competing interest.
Authors' contributions
CD carried out the pharmacological and qPCR assays, performed the statistical analysis and helped draft the manuscript. AH carried out sequence analysis and screened the primers. YN was involved in the pharmacological studies. YZ contributed to the study design and helped draft manuscript. GHL designed the study and drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by a grant to GHL from the National Natural Science Foundation of China (Grant No. 30972861).
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Deborah Guzzi is a healing facilitator specializing in Japanese Shiatsu and Reiki. She writes for Massage and Aromatherapy publications. She travels the world to expand her knowledge of healing and seeking writing inspiration. She has walked the Great Wall of China and visited Nepal (during the civil war), Japan, Egypt (two weeks before "The Arab Spring"), Peru, and France (during December's terrorist attacks).
Her poetry appears in Magazines: here/there: poetry in the UK, Existere - Journal of Arts and Literature in Canada, Tincture in Australia, Cha: Asian Literary Review, Hong Hong, China, Latchkey Tales in New Zealand, Vine Leaves Literary Journal in Greece, mgv2>publishing in France, and Travel by the Book, Ribbons: Tanka Society of America Journal, Emerge Literary Journal, and others in the USA.
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I've also heard in this thread that it sucks that Apple controls apps and treats developers like crap.
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- In the anime Elfen Lied, during her horrible childhood, a lonely Lucy meets Kohta and spends the summer with him. One of the most notable things they did was sitting out in the rain together while listening to the song "Lilium" coming from Kohta's music box. For the first time in her life, Lucy closes her eyes and is able to forget everything terrible that's happened to her thus far, for a moment. At various points during the series she hums the song, reveling in the wonderful memories she shared with Kohta. In her words, she "was able to survive that long because she clung to the hope that she would be able to meet Kohta again someday and apologize".
- In the earlier episodes of Neon Genesis Evangelion, Shinji regularly listened to Track 25 for "You Are the Only One" and Track 26 for "Blue Legend" on his SDAT player in an attempt to shut out the world. In episode 25 when the batteries finally go dead, he ignores it, which is a sign of his increased apathy.
- In Puella Magi Madoka Magica, while Kyousuke is in hospital with an injured hand, Sayaka tries to cheer him up by getting him to listen to classical music. It has the opposite effect, because it reminds him that he can't play the violin any more. Later in the series, when Sayaka becomes a witch out of despair after realising she can never be with Kyousuke, she has her familiars (who look like him) play music for her.
Fan Fiction
- In Racer and the Geek, Sunny sings a mix of Bronezhilet and Prishel Prikaz when remembering his past.
- In Songs Uncle Sings, also by Kalash93, the uncle sings such songs on the balalaika. At the story's climax, he and his nephew sing the first verse of Sineva.
- In Star Trek: First Contact, after being denied permission to take part in the defence of Earth, Picard stays in his office with Berlioz turned up so loud that objects on his desk vibrate.
- The 1989 Tony Danza vehicle She's Out Of Control had the teen protagonist lock herself in her room playing her music at full volume after a fight with her father and a breakup with her boyfriend. When one of her friends comes looking for her, she is directed "up the stairs, follow the sad music".
- Davy Jones' music box, a memento of his long-lost love, is like this in Pirates of the Caribbean. The melody can lull him into sleep or drive him to tears. The aforementioned long-lost love also has one, and it seems to have the same function for her.
- Edward in Twilight listens to music in his car to calm down after meeting Bella and almost killing her.
- In the Lonely Werewolf Girl books, Kalix likes to listen to the Runaways loudly when she is depressed or angsty (which is to say, often). Daniel does much the same with his Goth/Death Metal music whenever he feels angry or frustrated with Moonglow (again, often).
- In Ancillary Justice, the AI Hive Mind One Esk has a habit of singing and collecting songs. Later on after being reduced to a single body, One Esk often hums a particular song from her past in times of stress. Someone eventually sees through her disguise by hearing a song they recognize.
Live Action TV
- In Glee, lovesick school counselor Emma sings/cries along to Eric Carmen's "All By Myself" while sitting in her car in the rain.
- Xander plans to do this when Buffy rejects him in the first season of Buffy the Vampire Slayer.
"I'm just gonna go home, lie down, and listen to country music. The music of pain."
- In "Entropy" after Xander's wedding goes pear-shaped he gets drunk listening to "Sao Paulo Rain" by Tom McRae.
- In "Forever" Giles listens to "Tales of Great Ulysses" after Joyce's death, which he played for Joyce during the events of "Band Candy".
- "Lie to Me" reveals that when her friend Ford moved away Buffy would spend hours locked in her room listening to the song "I Touch Myself". She hastens to add that she had no idea what it meant at the time.
- In Angel, not only does the titular vampire listen to sad music when he's depressed, but his song of choice is Barry Manilow's "Mandy".
- A throwaway line in the Angel episode "Sanctuary" revealed that Angel tried to turn Faith on to the "healing power of the Manilow", and that while Manilow seriously wasn't Faith's thing, she had to admit she felt better sometimes after listening to "Weekend in New England"
- The Star Trek: Deep Space Nine episode "It's Only A Paper Moon" has Nog listening to "I'll Be Seeing You" loudly and repeatedly as he tries to get over his PTSD.
Web Original
- After the death of his teammate Jetstream, Achilles, leader of the Global Guardians, sat in his office with Peter Gabiel's "I Grieve" blaring at maximum volume. For seven and a half hours.
Western Animation
- In Lilo & Stitch, Lilo slumps on the floor and listens to "Heartbreak Hotel" after being rejected by the other girls.
- "Soon" is sung by Thumbelina to comfort herself because she's lonely. Her mother later sings it to comfort herself over her missing daughter.
- "Baby Mine" from Dumbo is sung by Mrs. Jumbo to comfort herself and her baby because they've been separated due to the former's going Mama Bear to protect her baby.


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Publisher's Description+
Simplify Media software lets you share your home library and connect with friends and family, right within iTunes. It works with Macs and PCs and MP3, WMA, AAC and Apple Lossless files. Give it a try. It is simple, safe and free.
This version add support for Apple Lossless files. Build 904 incorporate improvements related to external and network hard drives.
What's new in this version:
Build 903 adds support for Apple Lossless files.


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Baldwinsville A group of students from Van Buren Elementary School participated in the MVP Health Care 3K Fun and Fitness Run as part of the 19th Annual Syracuse Festival of Races on Oct. 2. This was the third year that Van Buren students stepped up to the challenge under the direction of the school's physical education teacher Daniel Vannatta. The run encourages students and their families to lead physically active lives. Students who took part in the race are (back, from left) Michael Allen, Luca Giannino, Cody Neish, Garrett Vannatta, Keaton Spindler, Carson Spindler, (front, from left) Natalie Doback, Myranda Dautrich and Jacob Neish. Missing is Owen Bauer.
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This volume has been a collaborative project from the start. It originated in meetings with Amy Beal, Eric Drott, and Peter Schmelz at the 2004 annual meeting of the American Musicological Society in Seattle. Over the next year, suggestions for further possible contributors were exchanged, and newly approached contributors in turn made their own suggestions. I am immensely grateful to all the contributors for their enthusiasm, their ideas, and their patience as the volume has taken its final shape. I hope they will be pleased with the outcome.
Our editor at Oxford University Press, Suzanne Ryan, has been just as influential a collaborator. She commissioned anonymous reviewers whose insightful reports helped to improve the volume immeasurably, and added her own encouragement and sound advice at many stages along the way. Her assistants, Norm Hirschy and Lora Dunn, have also been unfailingly helpful and efficient in their responses to my numerous queries.
Acknowledgments for permission granted to reproduce copyright material are included with the reproductions themselves.


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Watch out, it's the finnish axe murderer!
Great atmosphere btw.
From the depths, your journey home begins. Scintilla is about mystery, solving puzzles both of the gameplay and of the story, but there is also combat --and horror-- involved. Our themes come out of spiritual qualities, imagination, dreams, symbolism, and our goal is a vibrant, atmosphere-ridden adventure. This mod is being developed on Half-Life 2 Episode 2, and so it requires Ep2 to run.
Watch out, it's the finnish axe murderer!
Great atmosphere btw.
thats awesome :D i love the skybox
I'm loving all this glowy, blue stuff!
Depends. Lacunar, short-term, prosopamnesia..
I saw what you did there!
You need to find a bunch of gold ingots and craft a better axe, so you can break this gate.
not true. But that would be cool, now that I think of it.


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text: The Story of Barbaresco and the Feather. @Luisa_Rocca @winehouseLA Joanie Karapetian February 6, 2014 5 Comments My Lucky Day… I climbed the stairs to the tasting area at the Wine House yesterday morning for my appointment with the legendary Lance Montalto (Italian Wine Yoda), and had to do a double-take. There in front of me was this priceless lineup of Bruno Rocca wines, sitting casually on the bar. And in front of the bar were standing… Francesco and Luisa Rocca! Francesco and Luisa Rocca, flanking their importer Aaron Ainsworth. For normal people maybe this doesn't seem like an especially crazy occurrence. However, for an Italian Wine Geek, this is kind of like running into Bob Dylan at your corner coffee shop… or bumping elbows with Prince Charles at the checkout line while grabbing your groceries. Seriously. Italian Wine-making Royalty. Even after 10 years selling Italian wine, I admit I was more than a little starstruck! Francesco Rocca It was particularly emotional to taste the wines with Francesco guiding me through. The 2008 Rabaja was truly stunning, and the 2010s across the board are RIDICULOUS. These wines are all about finesse. Fragrant and nuanced- they smell like the stunning hillsides of Piemonte look. A feast for the senses. Elegant tannins, super high-toned minerality and punctuated acidity. They need time in the bottle to develop- they need time in your glass to open like the perfect rosebuds they are. God, I love Nebbiolo. Luisa Rocca Francesco and Luisa told me the story of the feather on the label (la piuma), which I have always loved to see on the shelves of various stores and wine shops. He said his father wanted a label that stood out form the rest- most of them being dominated by sketches of their estates, or of the rolling hillside vineyard landscapes common in Piemonte. Bruno Rocca also wanted a symbol for the enduring life of the wine in the bottle. A picture that could capture the endless preservation of that particular year in each bottle. What could be more permanent than the power of the written word? The feather- symbol for flight, and for the ancient Italian scribes- was the perfect emblem. Immortal Dante in Paradiso (Fresco by Philipp Viet) This in no small way reminds me of the allusion towards feathers and writers and immortality found in Dante's own name. Alighieri. Ali-ghieri. "Ali", meaning "wings" in Italian, not inconsequentially made of feathers- for me always represented both the author's ability to fly. To transport himself away from the mundane and the prosaic, towards eternity through his pen- also made from a single feather. If humanity's stretch towards eternity is possible with words, then why not with wine? Please Share this:Click to share on Facebook (Opens in new window)Click to share on Twitter (Opens in new window)Click to share on Google+ (Opens in new window)Click to Press This! (Opens in new window)Click to share on Pinterest (Opens in new window)Click to share on Reddit (Opens in new window)Click to share on LinkedIn (Opens in new window)Click to email this to a friend (Opens in new window)Click to print (Opens in new window) Related 5 Responses Francesca Maria February 6, 2014 Our Sicilian Corvo and Nero D'Avola are also very good wines. Reply Steve Haught February 6, 2014 Well said. Several years ago my wife and I were visiting the Bruno Rocca winery and had received a tour from a nice young lady. We sat down at a table to taste wine and much to our surprise a man walked in wearing black leather pants, several bracelets and took a seat next to me. I tried to be cool and hide my excitement but I was happy to meet Bruno Rocca. He was very friendly and thanked us for our interest in his wine. As we walked to our car I told my wife that I felt as if I had just met a rockstar. Indeed We had! Reply Duff's Wines February 6, 2014 I'm with you on Nebbiolo. Love it. It is somewhat overlooked in many discussions of the 'great' grapes. Not sure why when you can access reasonably priced efforts as well as some of the great Barolos and Barberescos at prices far less than fledgling California cab wineries deemed as 'cult'. Reply alden marin February 6, 2014 Well written, evocative and emotionally moving piece! Yes, nebbiolo is what even the best pinot noir wants to be, when it grows up!!!! : ) Reply Michael February 10, 2014 Great Pictures! Reply Leave a Reply Cancel reply


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LG G3 specs revealed by official LG site ahead of launch
With only a few hours before the LG G3 is officially unveiled, yet another leak has confirmed what specs the upcoming device will have by non other than LG themselves.
Watch Google I/O full keynote [video]
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Full Version of Angry Birds Now Available in Android Market
Enough messing around with getting the full version of Angry Birds. The highly-successful game is now available on the Android Market, which means you can get it directly from your


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Johnny Depp Admits He Wasn't Always Nice to Leonardo DiCaprio
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'MacGyver' Movie and TV Reboot: What We Know
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Foreign Language Classes
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Year long world language programs Pk-12
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FRENCH YOUTH PROGRAM FOR HOME SCHOOL – MORNING SESSIONS @ALLIANCE FRANCAISE ROSWELL Research shows learning a second language makes for stronger students overall. From the


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Just one block away, Northern Liberties was raging in all-out party mode, as young rock bands wailed to the crowds on Second Street and throngs of bargain shoppers swarmed the Piazza at Schmidt's edition of the Brooklyn Flea.
Inside Jerry's Bar nearby, at the corner of West Laurel and New Market Streets, meanwhile, the tranquil weekend brunch mood felt a world apart. A live guitar duo sent silky jazz standards into the gorgeously rebuilt bi-level space, its glowing white marble bar and dime-store tile floor trimmed with reclaimed wood doors sprouting craft beer taps. Diners forked into tawny brown pancakes striped with blueberry compote. Fluffy golden omelets came rolled around sweet, seasonal corn and house-smoked bacon.
"Did you try the homemade scrapple?" I asked my kids, urging them to break off a crispy chunk of the mysterious brown loaf, its cornmeal porridge infused with the piggy richness of shredded meat and pork stock. (They did, then fought me for the rest.) "Did you try the gravlax?" I asked, unfolding a translucent orange sheet of fennel-cured fish and draping it with the ripest slice of beefsteak tomato I'd seen this summer over a bagel with capers and cream cheese.
Slurrrp, went my straw, hitting briny bottom on my Bloody Jerry cocktail just as the guitar duo launched into a meandering riff on "Blackbird." Sigh. Do we have to leave into the blaring street-fair scrum?


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A Dodge County community collection to provide fresh produce to residents in need.
Bring in a donation of fresh produce to the Y August 10 through September 11 and receive a Family Day Pass to use or gift!
Produce donation must serve 4 or more; no spoiled or rotten produce will be accepted. All fresh produce items are accepted. Multiple donations are accepted with a limit of one Family Day Pass per day or donation.
Bring donation in bags or containers to the Welcome Center. Donations are only accepted Monday through Thursday as the produce is delivered daily.
Working together, we can ensure that more of our neighbors receive healthy and nourishing foods.


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Claflin will welcome its first Leader in Residence, Ambassador James Irvin Gadsden, on Nov. 6-8. Ambassador Gadsden will meet with University and student leaders, engage members of the Orangeburg community in international affairs, participate in selected classroom course settings and address the globalization of Claflin University with University leadership.
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I woke up, in my bed
In my pitch black room
My ghostly friend was back
And wanting to play
Her life was taken away
At a very young age
She wakes me
Night after night
Just to play
Like a child wants too
She was so young so pure
She didn't deserve the death
She had to adore
With a gunshot to the head
Just because
Her dad
Was a drunk
And didn't want to hear her cry
All scared of ghost of the night
And now
She is my ghost
In the night
And I welcome her in my home
I'm not afraid
Every soul needs a day
And I'm the one that makes her day
Night after night
And I don't think she will ever go away.
Ghostly Angel by valleighWatch
©2012-2016 valleigh
quite a different poem, and its so beautiful. you're a very talented writer.
despite lack of descriptive words, which no one necessarily needs for a poem, you managed to create some graet imagery/
i really love this poem.
despite lack of descriptive words, which no one necessarily needs for a poem, you managed to create some graet imagery/
i really love this poem.
This is a very different poem. I love the concept and it also conveys some emotions. the innocence of the girl, the contrast of ghost to angel and how you react to her and embrace her Love it!
thank you, a friend gave me the idea to write about a ghost and this is what popped into to my head
This is good if tragic though one little nit pick I think you mean endure not adore I could be wrong though
i mean what i said
Great development. Poor kid. Kids wit drunkard fathers have it hard.


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C Adams
Published in the Western Telegraph on 18 July 12
Mr Cecil James Adams Merlins Bridge (formerly of Robeston Wathen, Narbeth). The death occurred suddenly but peacefully at his home on Friday 6th July of Mr Cecil James Adams of Haroldston Mill, Mill Lane, Merlins Bridge, aged 81. He was the beloved husband of the late Barabara and dear friend of Betty. Funeral Thursday 19th July 2012 1.00pm at Parc Gwyn Crematoirum, Narberth. Family flowers only, donations if so desired for Robeston Wathen Church c/o Miss Mary Jenkins, 1 Beechwood Place, Narberth. Enquiries to Glyn Thomas & Son 01437 890571.
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Band Of Gypsies Chiffon Flyaway Vest
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- Lace back tie flyaway vest
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Today the NCAA announced in a press release that it has agreed to a new, 14-year contract with CBS and Turner to broadcast the NCAA men's basketball tournament.
(CBS, NCAA via SBJ's John Ourand: 96 Still In The Mix)
The release also addressed the size of the field:
Late Wednesday, the NCAA Division I Men's Basketball Committee unanimously passed a recommendation to the Division I Board of Directors to increase tournament field size to 68 teams beginning with the 2011 Championship. The recommendation will be reviewed by the Division I Board of Directors at its April 29 meeting.
That may lead some to believe that a 96-team tournament is off the table. But as confirmed by NCAA Interim President Jim Isch and CBS Sports President Sean McManus on a media conference call today, a 96-team field remains a possibility in the future - at the NCAA's discretion.
As I've noted here many times over the past three months, the decision on the size of the field is completely up to the NCAA Board of Directors. The new, 68-team tournament recommendation comes from hoops-centric NCAA membership, not the folks who will make the final call. The idea of a 96-team NCAA hoops tournament was never a consideration of NCAA basketball personnel.
The 96-team idea germinated from non-basketball NCAA representatives using the increased field size to raise TV broadcast rights fees, thus maintaining the proper staging of the nearly 100 annual NCAA championship events. Events which incur a massive, multi-million dollar shortfall on the governing body's balance sheet every year.
On April 29, I expect the NCAA Board of Directors to confirm that the 2011 NCAA men's basketball tournament will have 68 teams. That confirmation does not mean that a 96-team tournament is dead, only that the staging of a 96-team, 2011 tournament is now, according to a source, logistically impossible.
Today a prominent sports facility scheduling expert told me that if the NCAA suddenly wanted to book an increased number of dates at tournament sites for March 2011, it would likely be unsuccessful in that endeavor.
"It would be hard to get all the necessary arenas cleared and blocked for the games. They probably couldn't route it that fast."
2012 is another story.
What's most interesting about today's events is why the NCAA Division I Men's Basketball Committee made its 68-team recommendation in the press release knowing that it had no authority over the NCAA Board of Directors, which will make the final call on the size of the field on April 29. NCAA interim President Isch also maintained that 96 teams was still on the table during a subsequent media conference call.
Does that suggest that NCAA athletic directors, coaches and conference commissioners are not on the same page as the university presidents on the NCAA Board of Directors who hatched the 96-team plan?
How could you come to any other conclusion?
The NCAA's basketball arm is doing all it can to publicly wash its hands of 96, even if it means standing up to a Board of Directors over which is has no authority.
Though if anyone at this moment deserves credit for staving off 96 it isn't the NCAA basketball people. It's CBS.
The network brought in Turner to help increase rights fees on a property that it lost money on in 2010. Increased fees that McManus today confirmed apparently weren't predicated on 96 teams. CBS and Turner apparently also have no control on the size of the field beyond 68.
Unlike the university presidents on the NCAA Board of Directors, CBS smartly understands how wildly unpopular the 96-team prospect is, and apparently did whatever it took to prevent the major field expansion from immediately happening.
Though it does make me wonder had I not broke the 96-team NCAA tournament expansion story on Feb. 1, would we talking about 68 instead of 96 today?
It was that same story that NCAA vice president Greg Shaheen addressed specifically in his "done deal" denial to SI.com's Stewart Mandel shortly after my original piece.
While Shaheen appears to have won a battle, the war is far from over.


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