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During hibernation in the 13-lined ground squirrel, Ictidomys tridecemlineatus, the cerebral cortex is electrically silent, yet the brainstem continues to regulate cardiorespiratory function. Previous work showed that neurons in slices through the medullary ventral respiratory column (VRC) but not the cortex are insensitive to high doses of pentobarbital during hibernation, leading to the hypothesis that GABAA receptors (GABAAR) in the VRC undergo a seasonal modification in subunit composition. To test whether alteration of GABAAR subunits are responsible for hibernation-associated pentobarbital insensitivity, we examined an array of subunits using RT-PCR and Western blots and identified changes in ε- and δ-subunits in the medulla but not the cortex. Using immunohistochemistry, we confirmed that during hibernation, the expression of ε-subunit-containing GABAARs nearly doubles in the VRC. We also identified a population of δ-subunit-containing GABAARs adjacent to the VRC that were differentially expressed during hibernation. As δ-subunit-containing GABAARs are particularly sensitive to ethanol (EtOH), multichannel electrodes were inserted in slices of medulla and cortex from hibernating squirrels and EtOH was applied. EtOH, which normally inhibits neuronal activity, excited VRC but not cortical neurons during hibernation. This excitation was prevented by bicuculline pretreatment, indicating the involvement of GABAARs. We propose that neuronal activity in the VRC during hibernation is unaffected by pentobarbital due to upregulation of ε-subunit-containing GABAARs on VRC neurons. Synaptic input from adjacent inhibitory interneurons that express δ-subunit-containing GABAARs is responsible for the excitatory effects of EtOH on VRC neurons during hibernation.
- GABAA receptor subunit
- ventrolateral medulla
- ethanol
hibernating animals provide a natural model with which to investigate mechanisms of circuit-specific neuronal plasticity. During torpor [body temperature (Tb) = 4°C], the forebrain is isoelectric (54), and the dendritic processes of neurons in the cortex, hippocampus, and thalamus retract (51, 52). In contrast, neuronal control of respiratory function is maintained during torpor (34). Throughout hibernation, between torpor bouts (3–21 days), squirrels spontaneously rewarm (Tb = 37°C) and resume activity for <24 h. Previous work in our laboratory (22) has shown that during these arousals (interbout arousal) neurons in the ventral respiratory column (VRC) are insensitive to high doses of barbiturates. In contrast, cortical neurons are highly sensitive to the inhibitory effects of pentobarbital throughout the year. Similarly, VRC neurons in squirrels are sensitive to pentobarbital during the summer active months. Sensitivity to muscimol, a GABA receptor agonist, is unchanged in both VRC and cortex throughout the year, indicating the presence of fully functional GABA receptors. As barbiturates are potent allosteric positive modulators of GABAA receptors, circuit-specific alterations in GABAARs are likely to be responsible for the pentobarbital insensitivity in this medullary brain region during hibernation.
The VRC is a column of neurons extending through the ventrolateral medulla that encompasses the essential rhythm and pattern generating circuitry of the respiratory control system (2). The VRC consists of the Bötzinger region in the rostral medulla, the pre-Bötzinger complex (pre-BötC), and the rostral ventral respiratory group (rVRG) to the caudal ventral respiratory group (cVRG) in the caudal medulla. Identification of the VRC in squirrels is based on anatomical landmarks with reference to rats (37). Findings in squirrels suggest that medullary GABAA receptors are involved in preserving respiratory function during the extreme conditions that define mammalian hibernation (22). However, the specific molecular changes that take place in GABAARs in respiratory brain regions during hibernation are unknown.
GABAARs are pentameric and typically composed of α1(2)-, β2(2)-, and γ2-subunits. Two molecules of GABA bind the receptor at junctions of the α- and β-subunits, while the γ2-subunit facilitates postsynaptic localization (4). Thus far 19 GABAAR subunits have been found in nature (12). While the predominant GABAAR found throughout the rodent brain is the α1β2γ2-subtype, there are regionally localized subtypes that incorporate other subunits (39). GABAAR subtypes can help to define distinct neuronal circuits, subcellular localization, and pharmacokinetics, thereby contributing to unique circuit characteristics (41, 44). Subunit substitutions can radically alter cellular and network responses to GABAAR modulators. For example, when the δ-subunit is inserted in place of γ2, GABAARs are predominantly extrasynaptic and display increased sensitivity to allosteric modulators, especially ethanol (EtOH; Ref. 21), so much that they have been termed "a target for alcohol" (47). Less well understood is the ε-subunit, the function and natural expression patterns of which are largely unknown. Transfection of the ε-subunit results in GABAARs that are insensitive to pentobarbital, propofol, and benzodiazepines (13, 14, 23), yet a natural role for the ε-subunit has not yet been established.
There are three potential hypotheses to explain the previously reported pentobarbital insensitivity in VRC neurons during hibernation (22). First, there is insertion of ε-subunit-containing GABAARs into VRC neurons during hibernation. Second, there is a decrease in δ-subunit-containing GABAARs during hibernation. A third explanation as to how VRC neurons could become seasonally insensitive to pentobarbital is by changes in GABAA receptor subtypes on neurons that project to VRC neurons. Each of these hypotheses can be tested with molecular, electrophysiological, and pharmacological approaches. For example, insertion of δ-subunit-containing GABAARs on inhibitory interneurons projecting to VRC neurons would result in an excitatory response to EtOH. We used several different techniques to test these hypotheses, including quantitative RT-PCR, Western blot, immunohistochemistry, and multichannel recording from brain slices, recognizing the challenges associated with such studies in squirrel. Based on our findings, we propose a model of GABAAR distribution (involving receptors expressing ε- and δ-subunits) that explains how VRC neurons may become seasonally insensitive to lethal doses of barbiturate. These findings hint at a solution for the problem of how medullary networks involved in cardiorespiratory control remain active during hibernation while most other (higher) brain regions are functionally switched off for energy conservation.
Ethical approval.
All experimental procedures were in accordance with National Institutes of Health guidelines and were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee.
Experimental animals.
Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) were trapped in and around Madison, WI, between May and September. Animals were housed individually with access to food and water ad libitum. From May through September, animals were maintained at an ambient temperature of 22°C with a 12:12-h light-dark cycle [summer active (SA)]. In September-February, animals were housed in a dark room maintained at 4°C to facilitate hibernation. Seasonal hibernation is characterized by extended periods of time (3–21 days) spent in the torpid, hypometabolic state [torpor (T)], punctuated by a rapid return (∼2 h) to normothermia lasting for ∼12 h [interbout arousal (IBA)] before reentering torpor (10, 27). Food and water were removed after ∼2 wk of torpor/arousal cycles. During IBA, animals were active but did not eat or drink. All hibernating animals completed at least four full torpor bouts before being used in experiments. Torpor duration was monitored daily by the sawdust method (9). Briefly, animals were covered in sawdust upon entrance into torpor and checked every 24 h for either arousal (IBA) or a disruption of sawdust, indicating an arousal and reentrance into torpor since the last observation. Tb was measured with a rectal thermometer upon decapitation. Tb of torpid hibernators was 3–9°C, 35–38°C for IBA hibernators and 36–38°C for summer active animals. A total of 104 squirrels was used in the following experiments.
Tissue collection.
Brain tissue was collected from SA, T, and IBA animals. Animals (SA and IBA) were deeply anesthetized with isoflurane and killed by decapitation; T animals were placed in a cooled chamber and exposed to 5% isoflurane for 5 min before decapitation. The medulla (between C1 and the pons) was harvested. Cortical samples (CTX), including primary motor and somatosensory areas centered on a region equivalent to bregma −3.24 mm in rat (37), were harvested simultaneously (Fig. 1A). In a separate group of animals, tissues punches (diameter = 0.75 mm, depth = 0.75 mm; Stoelting, Wood Dale, IL) were taken from CTX, and two medullary regions previously reported to be seasonally insensitive to sodium pentobarbital, the VRC and the nucleus of the solitary tract (NTS; Ref. 22). Punches were taken from three slices (thickness = 750 μm) through the medulla inclusive of the BötC and extending through the cVRG (Fig. 1B). Tissue samples for Western blot were homogenized in buffer [65 mM Tris·HCl, pH 7.4, 0.108 M NaCl, and 0.05 M NaF, protease inhibitor (Roche, Indianapolis, IN), 2.57 mM EDTA, and 0.5% Triton X-100] and stored at −80°C. Total protein concentration of each extract was measured using a Nanodrop (Fisher Scientific, Waltham, MA).
Western blot.
Cortical and medullary lysates were subjected to SDS PAGE by separating 30 μg protein per lane on 12% gels. Proteins were transferred to 0.2 μm nitrocellulose membrane (Sigma-Aldrich, St. Louis, MO), blocked in Tris-buffered saline with Tween-20 (TBST; 50 mM Tris-HCl, pH 7.5, 15 mM NaCl, and 0.5% Tween-20) containing 5% Blotto (Santa Cruz Biotechnology, Santa Cruz, CA), washed in TBST, and incubated at 4°C overnight in TBST containing 1% Blotto and primary antibody [anti-GABAA δ 1:1,000, AB9752 (Millipore, Billerica, MA); anti-GABAA ε 1:500, 1:1,000, 1:5,000, ab35971 (Abcam, Cambridge, MA); anti-GABAA α1 1:1,000, PRB-564P (Covance, Emeryville, CA); and anti-GABAA γ2 1:1,000, PRB-569C (Covance)]. Subsequently, membranes were washed in TBST and incubated for 1.0 h in TBST containing 1% Blotto and secondary antibody (goat anti-rabbit or rabbit anti-mouse IgG 1:3,000; New England Biolabs, Ipswich, MA). Membranes were washed in TBST and incubated in enhanced chemiluminescent Western blotting substrate (ECL; Pierce, Rockford, IL) for 5 min. Chemiluminescence was detected by exposure to film (ISC Bioexpress, Kaysville, UT). Protein expression from crude homogenates was normalized to GAPDH (1:1,000 mms-580s; Covance) or histone 2B (1:1,000, ab45695; Abcam). A total of 51 animals were used for Western blot (24 for punches and 27 for whole medulla and CTX). A minimum of three samples from each behavioral state (SA, T, and IBA) was run on each gel, and all gels were replicated a minimum of 3 times with samples from different animals run on each replication.
Several antibodies to the GABAAR δ-subunit displayed nonspecific banding in Western blots of rat tissues (Santa Cruz sc-25705, Santa Cruz sc-31438, and Millipore AB9752). As there were no obvious differences in antigen affinity in squirrel tissues, we selected an antibody (Millipore AB9752) that has been used successfully in Western blots of rat hippocampus (28). For immunohistochemistry, we selected an antibody to the GABAAR δ-subunit raised in goat (Santa Cruz sc-31438) to colabel sections with antibodies made in rabbit and mouse.
Quantitative RT-PCR.
Cortex and whole medulla samples were collected as described above. Samples were stored at −80°C until homogenization in Trizol (Invitrogen, Carlsbad, CA). cDNA was synthesized from the extracted RNA with Moloney murine leukemia virus reverse transcriptase (Invitrogen). mRNA expression was normalized to GAPDH, the amplification of which did not vary significantly by behavioral state. Negative controls showed no detectable signal with any of the primer sets used, and each set displayed a single predominant peak in the dissociation curve at the expected melting temperature for each amplicon. The primer sequences were designed to span introns whenever possible to discount any product from genomic DNA. Primer specificity was assessed through NCBI BLAST analysis before use. Primer efficiency was tested through the use of serial dilutions. CT values from duplicate measurements were averaged, and relative expression levels were determined by the comparative CT method (26).
In the absence of sequence data from squirrels, PCR primer sequences were derived from rat and mouse sequences and tested for their ability to amplify cDNA from squirrels. DNA oligos were synthesized by Integrated DNA Technologies (Coralville, IA). The following DNA oligos recognized highly conserved sequences and amplified appropriately: GAPDH forward primer: 5′-ATGCCGCCTGGAGAAACC-3′ and reverse primer: 5′-GTAGCCCAGGATGCCCTTTAG-3′; GABAA α1 forward primer: 5′-ATCACAGAGGATGGCACCTTGC-3′ and reverse primer: 5′-TGGGCATCCATAGGGAAGTCC-3′; GABAA α2 forward primer: 5′-AGAACAACGCTTATGCAGTGGC-3′ and reverse primer: 5′-GTGGTTGCACTCTTGGAGATGG-3′; GABAA α3 forward primer: 5′-AACCGGGAGTCAGCTATCAAGG-3′ and reverse primer: 5′-TTGGGTGCCTGTATGCTTC-3′; GABAA α4 forward primer: 5′-GGTTTCTGCCAAGAAGGTACCC-3′ and reverse primer: 5′-TTTAAACAAACCGCCAGGCAC-3′; GABAA δ forward: 5′-GTCTGCCTGGTTCCATGATGT-3′ and reverse primer: 5′-GGAGGTGATGCGGATGCT-3′; GABAA ε forward: 5′-ACCTGAGCCTCAGCTGGA-3′ and reverse primer: 5′-GGT CCG AGG CTG TTG ACA-3′; and synaptophysin forward primer: 5′-CAGACAGGGAACACATGCAAGG-3′ and reverse primer: 5′-GGCCCAGCCTGTCTCCTTAAAC-3′.
Quantitative RT-PCR was performed with a Prism Sequence Detection System (model ABI 7000; Applied Biosystems, Foster City, CA). A total of 24 animals (8 each in SA, T, and IBA) was used for RT-PCR. All samples were run in duplicate at an annealing temperature of 60°C. After the final amplification cycle, a dissociation curve was generated to ensure that a single gene product was amplified. Samples were excluded on a well-by-well basis if contamination was evident when examining amplification plots. If samples were contaminated in control gene wells, the behavioral state mean for the control gene was used. By these criteria, only 2.2% of wells were excluded from the analysis.
To confirm that the PCR-amplified DNA was the correct molecular weight, gel electrophoresis was used to analyze the PCR product for a highly conserved subunit (GABAAR α1; Ref. 49). Samples from each behavioral state (SA, T, and IBA) and a DNA ladder (Promega, Madison, WI) were loaded on a 1% agarose gel (Bio-Rad, Hercules, CA) and run at 60 V for 2 h in a 1× Loenings buffer (4.36 mM Tris base, 15.44 mM NaH2PO4, and 902.6 μM Na EDTA). The gel was subsequently stained with ethidium bromide (3 μg/ml; Bio-Rad, Hercules, CA) for 10 min and destained for 30 min in double distilled H2O. Gels were photographed on an ultraviolet box (Fotodyne, Hartland, WI).
Animals from each behavioral state (4 each in SA, T, and IBA) were perfused with cold saline followed by 4% paraformaldehyde in PBS (pH 7.4). Fixed brains were cryoprotected with 30% sucrose and sectioned coronally (30 μm) on a freezing microtome. Eight matched sections were selected from the medulla of each squirrel at 60-μm intervals (equivalent to bregma −13.9 to −13.1 mm in rat; 36). This is the location of the rVRG and cVRG in rat. Three matched sections were selected from the primary motor and somatosensory cortex (centered on the equivalent of bregma −3.24 mm in rat). All medullary and cortical sections were reacted simultaneously. Sections were washed extensively in 0.01M PBS. After 1.0 h in blocking solution (10% normal donkey serum in 0.01 M PBS), primary antibodies were applied for 48 h at 4°C in blocking solution and 0.3% Triton X-100. Primary antibodies were used at 1:1,000: anti-GABAA ε (ab35971; Abcam), anti-GABAA δ (sc-31438; Santa Cruz Biotechnology), and anti-GAD67 (MAB5406; Millipore). Alexa-Fluor-conjugated secondary antibodies (568 donkey anti-goat IgG, 488 donkey anti-rabbit IgG, 647 donkey anti-mouse IgG; Invitrogen) were used at 1:300 in 1% normal donkey serum and 0.75% Triton X-100. Negative controls (2 medullary and 1 cortical section from each behavioral state) were reacted simultaneously with the omission of the primary antibody. Sections were mounted and coverslipped with Vectashield Hard Set mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). There were no labeled cells in negative control slices from all behavioral states.
Image acquisition and analysis.
All images were acquired during the same session using an Olympus Fluoview 500 laser-scanning confocal system (Tokyo, Japan) mounted on an AX-70 upright microscope. Images were analyzed using ImageJ software (W. Rasband, National Institutes of Health, Bethesda, MD). Images were scanned with different wavelengths sequentially to prevent bleed through. User-defined thresholds were applied uniformly to all images to measure the average pixel intensity, the number, and the area of particles. For quantitation, background fluorescence measured in negative control sections was subtracted from measurement of positive label. Data were normalized to the mean expression of label across all summer active sections. Images (see Figs. 1–9) were uniformly processed in Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) as follows: brightness levels adjusted, unsharp mask routine to improve edge detection, converted to 8-bit depth, and cropped.
To quantify the number of neurons immunopositive for both GABAAR δ and glutamic acid decarboxylase 67 (GAD67, a marker of GABAergic neurons) in the VRC and its periphery, two images (635 × 635μm) were taken in anatomical center of the VRC bilaterally, based on anatomical landmarks with reference to the rat (37). Three additional images were taken on one side dorsolateral to the VRC where we previously determined that the majority of GABAAR δ-expressing neurons were localized (see Supplemental Material, P1, P2, and P3 in Supplemental Fig. S1; Supplemental Material for this article is available online at the Am J Physiol Regul Integr Comp Physiol website). Two investigators, blinded to the identity of sections, counted double-labeled cells in each image (4 images/section, 8 sections/animal, and 4 animals/behavioral state). There was >95% concordance between the two investigators.
When appropriate, a two-way mixed ANOVA was used with a Tukey's honestly significant difference post hoc test to determine differences between regions and behavioral states. For nonparametric analyses, a Kruskal-Wallis rank sum test was used with subsequent post hoc pair-wise tests if the chi-square P value was <0.05.
A total of 17 animals (7 SA and 10 IBA) was used in electrophysiological studies. Electrophysiological experiments were conducted during the summer active months (May to July) and during naturally occurring IBAs (November to February), which ruled out temperature-dependent effects. Detailed methods can be found in Hengen et al. (22). Briefly, brains were removed and medullary and cortical slices (350 μm thick) were cut with a vibrating microtome (Campden Instruments, Layfayette, IN). Cortical slices contained primary motor and primary somatosensory areas while medullary slices contained the NTS and VRC (Fig. 1, A and B). Slices were placed into an interface recording chamber (Warner Instruments, Hamden, CT) and subfused with warm artificial cerebrospinal fluid (aCSF, 37°C) at a rate of 8 ml/min. Slices were maintained at 37°C by an automated temperature controller (Harvard Apparatus, Holliston, MA). The composition of the aCSF was as follows (in mM): 120 NaCl, 26 NaHCO3, 20 glucose, 2 MgSO4, 1.0 CaCl2, and 1.25 Na2HPO4. To increase the yield of spontaneously active neurons, aCSF containing 9 mM KCl was used in all experiments. Previously, we determined that the GABAergic contributions to spontaneous activity were unaffected by the increased KCl concentration (22).
Experimental protocol.
Spontaneous activity was recorded simultaneously from medullary and cortical slices from the same animal. Two 16-channel extracellular electrodes arrays (model a4 × 4–3 mm100–177; Neuronexus, Ann Arbor, MI) were placed in the VRC, slightly ventrolateral to the nucleus ambiguus (Fig. 1, A and B). One array was placed in the NTS, and one array was inserted perpendicular to the cortical layers, centered on layer 3. Slices were allowed to equilibrate in 9 mM KCl aCSF at 37°C with electrodes inserted for 2 h. Baseline activity was then measured for 1 h, followed by sequential application of increasing doses of EtOH (0.001, 0.01, and 0.1 M EtOH in aCSF) for 40 min each. To examine the contribution of GABAARs to the neuronal response to EtOH, a subset of slices were treated with bicuculline, a selective GABAAR antagonist, before EtOH application. Spontaneous activity was recorded from two medullary slices from each of three IBA animals. After equilibration, a baseline was established, followed by 60 min of 100 μM bicuculline application (Tocris Bioscience, Ellisville, MO). Slices were then treated with 100 μM bicuculline together with 0.1M EtOH.
Data analysis.
Raw data were processed as described in Hengen et al. (22). Individual neurons were identified using principal component analysis (1). Neuronal activity was averaged in 5-min bins and normalized to the mean firing rate during the 1-h baseline recording before drug application. Waveforms that were recorded on multiple, adjacent channels were counted only once. Neurons were discarded from analysis if their mean baseline firing rate was ≤0.01 Hz, if they were silent for >10 consecutive min during the 1 h baseline, or if their firing rate was consistently decreasing during the 1-h baseline. Individual bins were discarded if the absolute firing rate was >500 Hz or if traces exhibited evidence of mechanical disturbances (i.e., normalized firing rate increased and then decreased >100 SD from the baseline mean in <3 min). Based on these criteria, 12.7% of neurons and <1% of data bins were discarded.
To examine possible artifacts due to drift across the duration of recordings, time control experiments were run for 340 min with no EtOH application. Time control experiments were analyzed with Wald t-tests to test for an individual effect of time. For statistical comparison, a Wald t-test for individual effects, or a likelihood ratio test to examine overall significance of interactions was used considering time, dose, animal, neuron, and behavioral state (R Foundation for Statistical Computing, Vienna, Austria). Differences were considered significant if P < 0.05. The total number of neurons per behavioral state was used as the number of independent samples for relevant statistical tests and calculation of SE. All data are reported as means ± SE.
Since GABAA receptor subunit composition in squirrel has not been described previously, we initially confirmed that the commonly reported synaptic GABAAR subunits were present in CTX, NTS,and VRC of 13-lined ground squirrels across the year (SA, T, and IBA). Western blot analysis revealed that GABAAR α1- and γ2-subunits, which participate in ligand binding and synaptic localization, respectively, were expressed abundantly in all regions in SA, T, and IBA (n = 9 animals/behavioral state; data not shown). Identified bands migrated at 51 kDa (α1) and 46 kD (γ2), which agrees with previously described subunit expression in mouse, rat, and human (17, 24, 42).
Expression of GABAAR ε-subunit.
To determine if the seasonal expression of the GABAAR ε-subunit was consistent with pentobarbital insensitivity of VRC neurons during hibernation (22), we first examined transcriptional regulation of GABAAR subunits in the whole medulla and CTX. Cortical levels of mRNA for the ε-subunit were unchanged across the year (Fig. 2). In contrast, medullary mRNA for the ε-subunit was >3.5-fold greater in T than SA animals (Fig. 2). mRNA levels in the medulla during IBA showed a nonsignificant trend towards increase by comparison with SA animals (P = 0.16).
To confirm that regional and seasonal regulation of the GABAAR ε-subunit mRNA was reflected in protein expression, we examined cortical and medullary expression of the ε-subunit with Western blot (n = 9 animals/behavioral state examined, 3 animals/behavioral state shown). GABAAR ε-subunit protein was expressed robustly in the whole medulla yet was near the threshold for detection in CTX (SA, T, and IBA; Fig. 3A). Western blots of punches of the VRC (as opposed to whole medulla; n = 8 animals/behavioral state examined, 1 animal/behavioral state shown) showed GABAAR ε-protein in all behavioral states (Fig. 3A). Protein expression in the VRC was visibly increased during T by comparison with SA, consistent with the RT-PCR data (above) and the previously reported (22) pentobarbital insensitivity of VRC neurons during hibernation (Fig. 3A). The identified band migrated at 45 kDa, which is consistent with a synthetic peptide corresponding to human GABAAR ε. In the NTS, a cardiorespiratory brain region that shows partial pentobarbital insensitivity (22), GABAAR ε-protein expression was similar to that of VRC samples (data not shown).
Total protein concentration in equal sized punches from the VRC in T and IBA animals was greater than in SA animals (P < 0.001 and P < 0.005, respectively; see Supplemental Fig. S2). In contrast, protein concentration in equal sized punches from CTX and NTS did not vary across the year. In light of this inconsistent relationship in the VRC, normalizing to total protein for quantification of Western blots would introduce an error. Consequently, we turned to immunohistochemistry for protein quantification.
Expression of GABAAR δ-subunit.
An alternative explanation for seasonal pentobarbital insensitivity in VRC neurons due to changes in GABAAR ε-subunit expression is a decrease in δ-subunit-containing GABAARs, which, unlike ε-subunit-containing GABAARs, have increased sensitivity to allosteric modulators including barbiturates (33). Thus we examined mRNA for the GABAAR δ-subunit in the CTX and medulla to more critically evaluate transcriptional control of extrasynaptic GABAAR expression across the circannual cycle. In cortical samples, mRNA for the GABAAR δ-subunit was unchanged across the circannual cycle and, indeed, was the most stable of all six subunits examined (Fig. 2). In contrast, GABAAR δ-mRNA levels decreased significantly during IBA by comparison with SA (P < 0.05; Fig. 2). While this finding would be consistent with our second hypothesis, rates of transcription and translation can be affected by temperature changes during hibernation. Therefore, we used Western blot to examine protein expression in whole medulla as well as in punches of the VRC. While there was considerable variability among animals, δ-subunit expression increased robustly in the whole medulla during T and IBA by comparison with SA (n = 9 animals per behavioral state; Fig. 3B). Surprisingly, we were unable to detect δ-subunit protein in punches of the VRC in SA, T, or IBA (n = 8 per behavioral state; data not shown), suggesting that δ-subunit-containing GABAARs are expressed in the medulla but not specifically within the VRC. To reconcile the mismatch between medullary δ-subunit mRNA and protein expression during hibernation, we used immunohistochemistry for the δ-subunit using a different antibody (see below).
Expression of GABAAR α-subunits.
To confirm that the increases in GABAAR ε- and δ-subunit protein in the medulla were not the result of a general upregulation of GABAAR-subunits, we examined the expression of mRNA for GABAAR α1–4-subunits. Medullary GABAA α1–4-subunits did not change across the circannual cycle (n = 8 per behavioral state; Fig. 2). Taken together, the RT-PCR and Western blot data indicate that regulation of the GABAAR ε- and δ-subunits in the medulla and/or the VRC during hibernation differs greatly from the CTX and is not due to generalized changes in GABAAR subunit expression.
Synaptic alterations during hibernation.
Cortical, thalamic, and hippocampal neurons undergo dendritic retraction and synaptic degradation during each bout of torpor, followed by regrowth on arousal (51, 52). As many GABAARs are localized at synapses and as synaptic GABAARs contain α-subunits, we predicted that cortical GABAAR α-subunit mRNA would show evidence of this degradation and regrowth. Consistent with this, levels of mRNA for the α1-, α3-, and α4-subunits (Fig. 2; n = 8 per behavioral state) were significantly increased in cortical samples during T by comparison with SA animals, and mRNA for the α2-subunit similarly exhibited a nonsignificant trend towards increased expression (Fig. 2). In IBA animals, the increase in α1-mRNA compared with SA approached significance (176 ± 39% of summer active; P = 0.06; Fig. 2). No equivalent evidence for dendritic retraction and regrowth was detected in medullary samples. To address the issue of primer specificity, we confirmed the molecular weight of the PCR product recognized by our oligonucleotides against GABAA α1-subunit with gel electrophoresis (data not shown).
To further confirm that the transcriptional regulation of the α1-, α3-, and α4-subunits seen in CTX might be at least partially explained by state-dependent synaptic plasticity, we examined mRNA for synaptophysin (a presynaptic vesicle glycoprotein) in cortical tissues from SA, IBA, and T animals. Cortical synaptophysin mRNA increased more than sixfold in T compared with SA animals (P < 0.001), consistent with synaptic remodeling previously described (51, 52) in hibernating squirrels (Fig. 2). During IBA, cortical synaptophysin mRNA increased nearly 3.5-fold compared with SA animals (P < 0.005), and synaptophysin mRNA in CTX was significantly greater in T than IBA (P < 0.005). These data further underscore the absence of any change in cortical GABAAR ε- and δ-subunit mRNA during hibernation. We found no detectable changes in medullary synaptophysin mRNA across the circannual cycle (Fig. 2). Combined with the lack of change in GABAAR α-subunits, these data suggest that synapses in the medulla may be protected from hibernation-related retraction of dendrites and synaptic remodeling.
GABAA ε-immunoreactivity in VRC neurons changes with behavioral state.
Since state-dependent differences in total protein concentration in a fixed volume punch of the VRC made accurate quantification of Western blots difficult (Supplemental Fig. S1), we used immunohistochemistry to assess GABAAR ε-subunit expression in the VRC across the year. The predominant pattern of staining in the VRC was in the cytoplasm of neurons and their processes (Fig. 4). GABAAR ε-immunofluorescent signal intensity in the VRC of T animals was nearly double that of SA animals (P < 0.05; Fig. 4). Signal intensity in the VRC in IBA was intermediate between T and SA but not significantly different from either behavioral state (Fig. 4D). These immunocytochemical data confirm our observations in Western blots of punches through the VRC, that levels of GABAAR ε protein are greater in T than in SA.
The overall increase in GABAAR ε-immunoreactivity in sections through the VRC in T by comparison with SA animals was due to both an increase in the number of stained neurons (169%; P = 0.09) and an increase in the level of fluorescence in individual cells. There was a nonsignificant increase in the number of labeled neurons in IBA by comparison with SA animals (129.4%; P > 0.05). Mean signal intensity expressed as a percentage of SA intensity was as follows: IBA > T > SA (IBA = 104.2 ± 0.27% of SA intensity, n = 1,502 neurons; T = 102.3 ± 0.26% of SA intensity, n = 1,695; and SA = 100 ± 0.27%, n = 1,161; P < 0.001 for all comparisons). The size of GABAAR ε-immunoreactive neurons did not differ between behavioral states (P = 0.23).
To determine whether the increased number of GABAAR ε-immunopositive neurons during T and IBA by comparison with SA were concentrated in any part of the VRC, we examined eight matched sections through the VRC (4 animals/behavioral state). When data from the two VRC sample areas were combined, there was significant effect of region: the number of GABAAR ε-immunopositive neurons in one section through the rVRG (section 3, corresponding to bregma −13.3 in rat; Supplemental Fig. S2C) more than doubled in T by comparison with SA animals (T, 51.5 ± 7.6 neurons/section; SA 23.3 ± 5.9; P < 0.05).
We also quantified GABAAR ε-subunit immunoreactivity in the NTS of the same sections used for quantification of GABAAR ε-subunit in the VRC. Label in the NTS was punctate with very few immunoreactive neurons, and signal intensity did not change across the year (P = 0.52). Immunocytochemical data from sections through the CTX stained for GABAAR ε-subunit confirmed Western blot observations, as ε-immunoreactivity was nearly undetectable in cortical sections.
GABAA δ-immunoreactivity changes with behavioral state in the VRC.
Although Western blots indicated that δ-subunit expression increased robustly during hibernation in the whole medulla (T and IBA; Fig. 3B), we could not detect δ-subunit protein in punches of the VRC in any behavioral state, suggesting that δ-subunit-containing GABAARs are expressed in the medulla but not specifically within the VRC. Virtually no GABAAR δ-immunoreactive neurons were found in the VRC in any behavioral state, and no neurons were found double labeled for GABAAR ε- and δ-subunits. However, δ-immunoreactive neurons were consistently observed peripheral to the VRC, in particular dorsolaterally (Fig. 5). To determine whether the number of δ-immunopositive neurons dorsolateral to the VRC increased during hibernation, we examined eight matched sections through the rostrocaudal extent of the VRC (4 animals/behavioral state). Sections were also stained for GAD67 to identify GABAergic neurons. Very few GAD67-immunopositive neurons and no double-labeled neurons were found in the VRC in any behavioral state. No double-labeled neurons were found in any of the three sample areas dorsolateral to the VRC in SA animals. In contrast, in IBA and T animals, many double-labeled neurons were found dorsolateral to the VRG (Fig. 5). In IBA, a total of 17.3 ± 15.6 double-labeled neurons was found in images through three sample areas in eight sections per animal (P < 0.05, compared with SA) and in T, 31.8 ± 11.5 double-labeled neurons were found (P < 0.05, compared with SA).
Regional and seasonal effects of EtOH on spontaneous neuronal activity in VRC neurons.
As δ-subunit-containing GABAARs are highly sensitive to EtOH (20, 31), we recorded neuronal activity in brain slices containing the VRC in response to increasing doses of EtOH in hibernating (IBA) and SA animals. Neuronal activity from the multichannel electrode array placed in the region of the VRC was initially categorized as follows: rhythmically bursting (<10%; Fig. 6A), fast tonic (∼60%; Fig. 6B), and slow tonic (∼40%; Fig. 6C). During the equilibration period, all rhythmically bursting cells converted into tonically active cells.
In slices from SA animals (n = 4 animals), increasing doses of EtOH (0.001, 0.01, and 0.1 M) failed to alter the spontaneous firing rate of neurons in the VRC (n = 33 neurons) (Fig. 6D), consistent with the absence of δ-subunit protein expression in Western blots and the absence of δ-immunoreactive neurons. All three doses of EtOH increased the firing rate of VRC neurons (n = 70 neurons) in slices from IBA animals (n = 4 animals) three- to eightfold (by comparison with SA animals, P < 0.05; Fig. 6D), consistent with the expression of δ-subunit-containing GABAARs on inhibitory neurons that modulate VRC activity.
In the NTS (n = 28 neurons), the two lower doses of EtOH failed to alter the activity of neurons in IBA animals by comparison with SA animals (Fig. 6D). There was, however, a significantly different response to 0.1 M EtOH (n = 28 neurons; Fig. 6D). In CTX, all three doses of EtOH reduced the firing rate of neurons in SA animals (n = 57 neurons). Cortical neurons in IBA animals showed variable responses to EtOH (n = 57 neurons; Fig. 6). In response to 0.01 M EtOH, cortical neurons in IBA animals increased activity by 83 ± 37%, whereas cortical neurons in SA animals showed reduced activity (30 ± 16%; Fig. 6F). There was no significant effect of time on spontaneous neuronal activity in the VRC (n = 49 neurons) or the NTS (n = 36 neurons) in time-control experiments (3 IBA and 3 SA). Cortical neurons (n = 48 neurons) displayed higher variability in spontaneous activity, yet bursts in activity (<2 min) during time control experiments were transient and considered to be of no biological significance.
The excitatory response of VRC neurons in IBA animals to increasing doses of EtOH (Fig. 6D) was due to the activity of a subset of neurons (25 of 88 neurons). The majority of VRC neurons were nearly silent in baseline conditions (60 min). Neurons with baseline activity of <0.02 Hz were excited by the application of increasing doses of EtOH (Fig. 7A; P < 0.005, baseline compared with 0.1 M EtOH). Neurons with baseline activity between 0.02 and 0.5 Hz were variably affected by the increasing doses of EtOH (Fig. 7B; P = 0.07), while nearly all neurons with baseline activity >0.5 Hz showed a trend towards inhibition at increasing doses of EtOH (Fig. 7C; P = 0.09). These data indicate that mean network activity in the VRC is driven by a subset of neurons but that the majority of VRC neurons are either unchanged or inhibited by EtOH.
GABAAR contribution to excitation by EtOH.
To confirm that GABAARs are integral to the excitation of VRC neurons by EtOH, slices from IBA animals (n = 3) were pretreated with 100 μM bicuculline before the application of 0.1 M EtOH. Following 1 h of bicuculline, 0.1 M EtOH and 100 μM bicuculline were coapplied for 1 h. There was no difference between spontaneous activity at steady state in bicuculline and spontaneous activity in the presence of 0.1 M EtOH and bicuculline (133 ± 13% of bicuculline steady state; Fig. 8). Thus the GABAAR antagonist bicuculline prevented the excitation of VRC neurons elicited by 0.1 M EtOH alone (P < 0.05).
We have identified hibernation-associated changes in GABAAR subunits in neurons in the medulla that are not evident in the cortex of the 13-lined ground squirrel. These changes are consistent with a previous description of seasonal pentobarbital insensitivity localized to the VRC (22). Specifically, seasonal expression of the GABAAR ε-subunit in VRC neurons prevents inhibition by pentobarbital and EtOH during hibernation states (T and IBA). Synaptic input from adjacent inhibitory interneurons that express δ-subunit-containing GABAARs further prevents inhibition of VRC neurons by pentobarbital and EtOH. A proposed model of hibernation-associated regulation of GABAAR subunit expression in the ventrolateral medulla in the 13-lined ground squirrel is shown in Fig. 9. In light of these data demonstrating a critical role for GABAARs in hibernation, it is difficult to ignore the possibility that hibernation states (IBA and T) may be ligand driven. If so, this model illustrates a solution to the problem of how medullary networks involved in cardiorespiratory control remain active during hibernation while most other (higher) brain regions are functionally silenced for energy conservation.
A key feature of the proposed model (Fig. 9) is the inhibition of GABAergic inhibitory interneurons due to δ-subunit-containing GABAARs on their surface. Extrasynaptic GABAARs, including those containing δ-subunits, are highly sensitive to ambient levels of GABA from adjacent synaptic transmission, as well as being the primary targets for allosteric modulators such as pentobarbital (19). The proposed model makes very specific predictions with respect to recording from VRC neurons following application of EtOH to a slice preparation: neurons should show a net excitation. Had the expression of GABAAR-expressing δ-subunits been on noninhibitory neurons in this network, the VRC would have been inhibited by EtOH. Likewise, had the expression of GABAAR-expressing δ-subunits merely decreased during hibernation, there would have been no GABAAR-dependent effect of EtOH.
Experimental limitations.
Hibernation provides a unique opportunity to study naturally occurring mechanisms underlying regionally and seasonally regulated neuronal activity. However, there are significant challenges and limitations associated with studies in the wild-caught, 13-lined ground squirrel. First, specific activity states can only be studied at certain times of year. Second, genome-specific primers and species-specific antibodies are not currently available. Consequently, in this study we used a range of molecular and immunohistological techniques to identify the mechanism(s) responsible for pentobarbital insensitivity in the VRC during hibernation that was described previously (22). Each method builds on and supports the original observation that cardiorespiratory neurons are insensitive to the effects of lethal doses of barbiturate via plasticity of expressed GABAAR subtypes. Predictions of the hypothetical model (Fig. 9) were tested with electrophysiological and pharmacological approaches that avoid species-related issues (GABAA receptors: bicuculline and muscimol; ε- and δ-subunits: pentobarbital and EtOH). The most disparate respiratory states are SA and T, and we find a significant difference between GABAAR ε-subunit immunoreactivity in the VRC in these two behavioral states. We also found a trend towards increased GABAAR ε-subunit expression in the VRC in IBA by comparison with SA, and the electrophysiological data enabled us to unambiguously confirm the functional presence of barbiturate- and EtOH-insensitive GABAARs during IBA compared with SA. Taken together, there is compelling evidence for the seasonal insertion of GABAAR with ε- and δ-subunits in the VRC.
Temperature-related confounds (40) were avoided in this study by the inclusion of IBA animals (Tb = 37°C). We found greater total protein concentration in Western blots of equal sized punches of the VRC in T and IBA animals by comparison with SA animals, which was not seen in punches of CTX and NTS. We speculate that this hibernation-associated difference in total protein per unit volume of VRC tissue may be related to the maintenance of synaptic connectivity necessary for respiratory function at low temperatures in this brain region.
Identity of neuronal networks and recorded neurons.
We cannot say with certainty that the neurons we recorded in the VRC are definitively respiratory. The anatomy of the squirrel medulla is very similar to the rat, and placement of microelectrode arrays was restricted to a region comparable to the rat VRC. Furthermore, neurons with properties characteristic of previously described respiratory neurons (rhythmic bursting; Fig. 6A) were identified at this site (2, 3, 8, 45, 18). Previous studies (15) of brainstem networks controlling phrenic motoneurons show third-order neurons that project to premotor neurons in the VRC. The location of these neurons overlaps with the region dorsolateral to the VRC in which we found neurons immunopositive for the δ-subunit during hibernation (P1, P2; Supplemental Fig. 2; Fig. 9), many of which were colabeled with GAD67, a marker of GABAergic neurons. This region in the rat is also rich in calbindin and parvalbumin-immunopositive neurons, which are frequently associated with GABAergic function (36, 2, 3). GABAergic transmission plays a key role in respiratory neuron excitability (57, 43). As there are few GABAergic neurons within the VRC (55, 36), one of the sources of GABAergic input is likely to be this population of third-order neurons dorsolateral to the VRC.
While we cannot identify the specific role of any of the neurons recorded (i.e., rhythm generating, premotor, etc.), a majority of neurons in the VRC appear to be involved in the control of breathing (2, 3). Our finding that in IBA 25/88 of VRC neurons are excited by EtOH suggests that GABAergic/δ-subunit-expressing neurons normally inhibit the activity of a subset of VRC neurons and that this inhibition is relieved by the application of EtOH (Fig. 9). Thirty-seven neurons were unaffected by EtOH, suggesting that they did not receive input from GABAAR δ-subunit-expressing neurons or that these inputs were severed in the slice preparation. We also found that 0.1 M EtOH application to medullary slices from IBA squirrels produced a transient yet significant excitation of the NTS, a medullary region that is also critical to cardiorespiratory function. The NTS lies adjacent to a region containing GAD67/δ-immunoreactive neurons and may also receive input from δ-subunit-expressing GABAergic neurons similar to the VRC (see P3, Supplemental Fig. S2).
Role of GABAA receptors in respiratory control.
GABAergic neurotransmission plays an important role in the control of breathing (30, 43, 38, 25, 57). Glycinergic neurotransmission is responsible for reciprocal inhibition of expiratory neurons in the pre-Bötzinger complex, which contains the respiratory rhythm-generating network, and the excitability of expiratory neurons is modulated by both GABAA and glycine receptors (43). Additionally, inspiratory neurons in the VRC are tonically inhibited during the expiratory phase of breathing primarily by activation of GABAARs (25). Zuperku and McCrimmon (57) presented compelling evidence for a dynamic, nonlinear mechanism of gain modulation in the respiratory control system mediated by GABAergic neurons and GABAA receptors. This gain modulation allows the respiratory rhythm to be maintained while increasing or decreasing the output of the system when conditions change, such as during exercise, development, weight gain, hypoxia, or aging (57). Hibernation imposes a significant challenge to the respiratory control system for gain modulation to maintain normal blood gas levels. The location and balance of GABAARs expressing ε- and δ-subunits are thus potentially very important to the functioning of respiratory networks, as both subunits are capable of forming extrasynaptic ligand channels that can provide tonic mediation of cell excitability, independent of synaptic transmission (48, 53, 5). Recent preliminary data in rats indicate that there are GABAARs expressing ε- and δ-subunits in the ventrolateral medulla and that the responses of VRC neurons to pentobarbital are similar to those of SA squirrels (K. Hengen, unpublished observations). It will be interesting to determine whether there are changes in the expression of these subunits in response to demands for enhanced gain modulation in respiratory control.
GABAAR stoichometry.
To our knowledge, these are the first data to support the existence of naturally expressed, pentobarbital-resistant GABAARs, initially described in transfected HEK-293 cells and formed by α(2)β(2)ε-subunits (13). Because normal ligand binding requires two α and two β-subunits, these data and our previous work suggest α(2)β(2)ε receptors lack a γ2-subunit. Evidence suggests that GABAARs receptors lacking a γ-subunit are primarily extrasynaptic (4); thus the ε-subunit-containing GABAARs described here in the VRC may be extrasynaptic. This will need to be confirmed in future studies, as a recent report of ε-subunit-containing GABAARs in the locus coeruleus of rats suggested that these receptors were pentobarbital sensitive and located perisynaptically (7). In light of this finding, knowing the identity of the other four subunits will be important, as they may contribute to the affinity of allosteric modulators.
Biological significance.
The hypothetical configuration of GABAAR subtypes in the VRC (Fig. 9) could explain the insensitivity of hibernating squirrels to a wide variety of anesthetics that act on GABAARs (13, 14, 23) including lethal doses of pentobarbital (35). Pentobarbital, propofol, and other anesthetics interact with VRC neurons, at high doses abolish neuronal activity in medullary slices (11), and inhibit neuronal control of respiration in vivo (16, 56). EtOH, pentobarbital, and other anesthetics have limited natural or evolutionary relevance, but these compounds act on GABAARs in a manner similar to that of neurosteroids (53, 46, 6, 19). Furthermore, during pregnancy when neurosteroid levels are greatly elevated, GABAARs containing the δ-subunit are upregulated (28). We speculate that circannual changes in the levels of neurosteroids in hibernators underlie the reorganization of GABAARs such that forebrain networks are silenced while respiratory function is maintained. On entry into torpor, this pentobarbital-like ligand, in combination with temperature-dependent cessation of neuronal excitability below 15°C (54, 50), would silence all neurons capable of binding this ligand. Only by means of a regionally restricted ligand binding capacity could neurons in the forebrain be silenced and those in the medulla remain functional. Nonetheless, ligand insensitivity in the medulla would not prevent the temperature-dependent retraction of neuronal processes described previously in cortex (51, 52, 40). While there is compelling evidence that the forebrain undergoes morphological remodeling in torpor, our data suggest that the medulla does not. Our analyses of cortical synaptophysin mRNA are consistent with the report by von der Ohe et al. (51, 52) that up to 65% of synapses are degraded and reformed in a temperature-dependent manner. However, synaptophysin mRNA did not change in the medulla, which appears to be spared from synaptic degradation during torpor. We hypothesize that the increased total protein concentration in the VRC during hibernation states (IBA, T; Fig. S1) may represent the upregulation of a factor responsible for preventing the synaptic retraction observed elsewhere in the brain. Determining whether this change in total protein concentration is due to alterations in intracellular scaffolding proteins, extracellular matrix proteins, or glia will provide further insight into mammalian survival at low Tb.
In conclusion, changes that the nervous system undergoes during hibernation may differ greatly by region. Our data suggest that, during hibernation, medullary regions controlling breathing are protected from the temperature-dependent synaptic remodeling that occurs in the cortex. Alterations in GABAA receptor ε- and δ-subunits on distinct populations of neurons in the VRC may provide a network that permits breathing and survival at Tb < 4°C. These findings in hibernators advance our understanding of the impact of barbiturates and EtOH on the respiratory control system.
This work was supported by the National Institutes on Aging Grant AG-18760 (to M. Behan). K. Hengen was supported by T32 GM007507.
No conflicts of interest, financial or otherwise, are declared by the author(s).
We acknowledge input from Nathan Nelson and Drs. Hannah Carey, Adrianne Huxtable, Mathew Jones, Clark Nelson, and Thomas Tubon. We thank Dr. Cecile Ané for assistance with statistical modeling.
- Copyright © 2011 the American Physiological Society


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unauthorized check dam construction at Idukki - 34 mins ago
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The last male northern white rhino in the planet
Sudan is the only male of the world's last three remaining northern white rhinos.
As its name suggests, the animal was found in 1980s in Sudan, which, together with DR Congo and Central African Republic, used to be the species' habitat.
However, years of poaching for rhino horns as well as conflicts in the region have resulted in the animal's disappearance from the wild almost a decade ago.
Sudan had been kept at a zoo in Czech Republic before it was brought to the Kenyan conservancy in 2009 with another male and two females, in a hope that here can provide "the last chance of survival" for them and help them breed.
Unfortunately, at 43, Sudan is too old to mate as the animal usually has a life expectancy of 40 years in wild.


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Stranded travellers on Barbados holidays still enjoying "Good Barbadian hospitality"
Despite the fact that many British visitors are still stranded in their never-ending Barbados holidays, many have received a comforting pat on the shoulder by the Barbadian Tourism Minister who's been spreading Barbadian hospitality among stranded guests.
As many as 3000 British travellers are still stranded in Barbados unable to put an end to their Barbados holidays – and all because of the spread of Icelandic volcanic ash that has grounded British-bound flights. Even when as of yesterday some long-haul flights resumed operations again, the Barbados Hotel & Tourism Association (BHTA) also stated that about 800 travellers were still stranded across various Barbados hotels.
Barbadian Minister of Tourism Richard Sealy gave these numbers last Tuesday morning as he addressed Britons booked in for longer-than-expected Barbados holidays at the Almond Beach Club & Spa. The minister said his visit to Almond was one of the many he had undertaken "to interface and empathise" with British holidaymakers who could not readily leave for home.
And it looks like it's not all gloom and doom for those still stuck on their Barbados holidays as Sealy added that the guests he had met so far had been "very understanding, to the point that they are volunteering to extend their vacations".
He hailed Virgin Atlantic and British Airways' efforts for providing accommodation and other comforts for "the stranded guests" for the longer duration of their stay in Barbados hotels. And, while the minister was quick to express his joy over flights - though limited – having resumed operations in Europe, he also express his concern about "suggestions of further activity in Iceland", which he reassured, the Government of Barbados was "closely monitoring".
"We are trying to avoid having crowds at the airport waiting to get on the plane, and people having to sleep at the area in front of the check-in counter," Sealy added.
Making clear that his activities visiting stranded British holidaymakers at their resorts didn't mean to interfere with or overshadow the airlines' many endeavours to look after their stranded passengers, Mr Sealy noted: "I am not in any way trying to usurp the role that the airlines are playing . . . . It is their responsiblity; but we are partners."
Matthew Clayton from Mumbles, South Wales, who should have left last Friday, said that despite the inconveniences he was enjoying his longer Barbados holidays and was extremely happy with the hospitality he was receiving at Almond Beach Club & Spa, but unhappy that he and his wife Carolann did not know when they would be able to go home.
"My wife is the deputy headteacher at a large school . . . . She is away and the headmaster is stranded in Spain. While I am a self-employed consultant and can do my work from here, my wife will not be paid for the time she is spending here" - said Clayton.
On the other hand, hotel director Gerry Lewis said Almond Beach Club & Spa was constantly in contact with the airlines and tour operators, as the primary objective was to ensure the British visitors were comfortable and were given as much as information as became available so they could make the best of their forcefully-extended Barbados holidays.
Lewis added that like many other Barbados hotels faced with same situation, Almond Beach Club & Spa's aim was that the British "enjoy good Barbadian hospitality while they await their departure".
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United States District Court Eastern District of Michigan
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1 United States District ourt Eastern District of Michigan FILING YOUR LAWSUIT IN FEDERAL OURT Prepared by the Office of the lerk United States District ourt 231 W. Lafayette Street Detroit, MI
2 REV. 7/01 The Different Kinds of ourts What kinds of cases are filed in U.S. District ourt? You may be aware that there are both federal courts and state courts. In Michigan, state courts are divided by county, and enforce state and municipal laws. Each Michigan county has a ounty ircuit ourt, which decides issues brought under the laws of this state. That includes family disputes, probate, broken contracts and matters brought under the State onstitution. Also, every city has a District ourt, which decides municipal matters (traffic tickets, landlordtenant, zoning, etc.) Although the Michigan state courts must enforce the federal onstitution and laws, most of the cases they decide involve the constitution and laws of the state. Federal courts are established by the U.S. government to decide disputes concerning the federal onstitution and laws passed by ongress. Our state has two federal courts, the Eastern District of Michigan and the Western District of Michigan. The Eastern District covers the eastern half of the lower peninsula. The Western District covers the rest of the state. There are three types of cases filed in U.S. District ourts: 1. ases where the United States government is a party. Lawsuits for social security or veterans benefits are among those that would be filed in a federal court. Also, cases against any federal agency, like the Postal Service or Internal Revenue Service. 2. ases brought under federal statutes. U.S. District court is limited to hearing the specific types of cases described in the onstitution or specifically provided for by ongress. Federal laws can cover issues not addressed by state laws, such as interstate commerce, damages at sea, labor laws, environmental matters, agriculture, certain tax matters and many other laws. Also, federal laws can duplicate state laws, such as in civil rights protections. 3. ases where the parties reside in different states. These types of matters are called diversity cases. In a diversity case, if you live in Michigan and you are suing someone in another state, the Judge will decide which U.S. District ourt is most appropriate to hear your case. This is called the venue. In deciding the proper venue, the judge will consider where the case was filed, where the majority of the parties are located, where the witnesses reside, where the actual damage was done and other issues. A rule of thumb is that the case will be heard at the ourt closest to the defendant. But the judge has a lot of latitude. Diversity cases must claim a minimum of $75,000 in damages. If your 1
3 Before you file, please consider: Should I file in State ourt or Federal ourt? Rule 11 of the Federal Rules of ivil Procedure prohibits the filing of lawsuits that are clearly frivolous or filed just to harass someone. If the court determines that you have filed a lawsuit for an improper or unnecessary reason, it may impose sanctions against you, including ordering that you pay any legal fees of the party that you sued. If you are suing someone based on a violation of a federal statute, for example the Americans with Disabilities Act, you would file your case in U.S. District ourt. However, state laws and federal laws sometimes overlap each other. For example, Michigan s Elliott-Larsen ivil Rights Act offers protections to citizens similar to federal civil rights statutes. In other words, you can file a civil rights case in either federal court or Michigan state court. To choose which court would be best, you need to look at other things: Where will the jury come from? How long will it take for your case to get to trial? What are the fees involved in each court? In State court, your jury will be picked from citizens residing in the county where the case was filed. In federal court, the jury will be selected from a pool of people who reside throughout the eastern half of the lower peninsula. Also, the typical U.S. District ourt case takes about a year to litigate. Michigan s state courts have different timelines. It may be worth your time to do some research. 2
4 ATTORNEYS You have the right to represent yourself. You do not have to have an attorney. If you are representing yourself, the ourt may show you some extra consideration. The Judge will still expect you to state your case clearly, to make all your deadlines, and follow our filing procedures. If you can afford to hire your own attorney, but don t know of any, you might consider contacting the Detroit Bar Association s Referral Service. That program will tell you which Bar Association members practice the type of law that you need help with. The number to the Detroit Bar Association is Where can I do legal research? Wayne State University, the University of Michigan and the University of Detroit-Mercy Law School libraries are open to the public, although they will not let you check out material unless you are a student. Some college libraries have law-related collections, including Henry Ford, Oakland and Macomb ommunity olleges; Oakland and Madonna Universities and Walsh ollege. These collections are not as extensive as you would find at a law school library. The public libraries in Dearborn, Detroit, Farmington, Flint, Southfield and Port Huron maintain law-related collections, too. Will the ourt appoint a lawyer to represent me? If you would like an attorney, and cannot afford to hire one, the ourt may appoint an attorney to represent you. To ask the ourt to appoint a lawyer, please fill out an Application for Appointment of ounsel form, available in the lerk s Office. The Judge bases the decision to appoint an attorney on two items: your financial situation and the merits of your case. Where can I get free legal advice? If you cannot afford an attorney, but need legal advice, there are a number of agencies that can help you: Detroit Legal Services linic , Ext. 206 Immigration Legal Services/Archdiocese of Detroit Lakeshore (Macomb ounty) Legal Aid Port Huron Office aro Office Legal Services of Eastern Michigan Bay ounty Office Genesee ounty Office Midland ounty Office Saginaw ounty Office Legal Services of Southern Michigan (Jackson) Monroe/Lenawee ounty Office Washtenaw ounty Office Legal Hotline for Michigan Seniors Oakland/Livingston Legal Aid (Pontiac) Howell Office Salvation Army Legal Aid linic State Bar of Michigan Lawyer Referral Service For counties with no local referral service program University of Detroit-Mercy Urban Law linic University of Michigan Law School linic Wayne ounty Neighborhood Legal Services
5 How do I file a lawsuit? If you have decided to file your lawsuit in U.S. District ourt, you will need to file a complaint in the lerk s Office. What is the filing fee? an it be waived? The fee to file a complaint is $ There are no other fees involved. If you do not have the $150.00, you may apply to have the fee waived. Ask the lerk for an Application to Proceed Without Prepayment of Fees. That form is a financial affidavit, where you list your income and expenses. What is a complaint? The complaint is a document telling the judge who you are suing, what your case is about and what you want the court to do about it. You may write your own complaint, or use a form available in the lerk s Office. Make your complaint clear and understandable. This is the time to go into as much detail as you can. Don t be overly brief, and expect to tell the Judge the details of your case later. Do it now. The judge can dismiss your case if you do not go into detail. The Judge has to understand what your issues are. Along with the complaint, you will need to fill out some other forms, which you can get from the lerk s Office: 1) A ivil over Sheet, which asks some statistical information about your case. Directions for filling out a civil cover sheet are on page 7. 2) A summons, a form that lets people know that you are suing them. Directions for filling out a summons are on page 8. The above forms must be filed with any lawsuit. Some additional, voluntary forms are: 3) An Application to Proceed without Prepayment of Fees, if you are unable to pay the filing fee of $150.00, and 4) An Application for Appointment of ounsel, if you wish the court to appoint a lawyer to represent you. When your paperwork is completely filled out, file it with the lerk s Office. 4
6 When you re ready to file your complaint When you have your paperwork ready, please come to the lerk s Office on the fifth floor of the Theodore Levin U.S. ourthouse in Detroit. (Or, if more convenient, you may file at our offices in Ann Arbor, Flint or Bay ity.) U.S. District ourt offices: 231 W. Lafayette Fifth Floor Detroit, MI (313) E. Liberty Room 120 Ann Arbor, MI (734) Washington Avenue Room 304, P.O. Box 913 Bay ity, MI (989) hurch Street Room 140 Flint, MI (810) What happens when I come to the lerk s Office? When you come to the lerk s office, you will need to bring with you the following paperwork: 2 civil cover sheets, 2 summonses for each defendant, 2 complaints PLUS 1 copy for each defendant (So, if you are suing two people, you would need four copies, total) 2 Applications to Proceed without Prepayment of Fees IF you are asking the court to waive the $ filing fee, or the filing fee, if your are not asking the court to waive fees, and 2 Applications for Appointment of ounsel IF you are asking the court to appoint a lawyer to represent you. The clerk assisting you will review your paperwork to make sure we have everything we need. He or she will make sure that all of the questions have been answered on the forms and that you have enough copies. If everything is in order, and you are paying the filing fee, the clerk will: file-stamp your documents, assign a case number and judge, sign your summons forms, and write you a receipt for the $150 filing fee If you are asking that the filing fee be waived, the clerk will: stamp received on your complaint (it cannot be marked filed until the judge agrees to waive the fee), and assign a case number and judge The clerk will then send the paperwork to the judge, who will rule on whether or not to waive the fee. If the judge waives the fee, the clerk will then filestamp your documents, sign the summonses and, if the judge orders, arrange for the complaint to be served on the defendants. If the judge declines to waive the fee, the clerk will mail all of your paperwork back to you, with a letter saying that, if you would like the case filed, you will need to pay the filing fee. 5
7 How Do I Fill Out the ivil over Sheet? The ivil over Sheet asks some statistical information about your case. It looks much more complicated than it is, because of all the options you can check off. It is broken down into eight sections: Section I a. asks the name of the plaintiff and the defendant. b. asks for the counties where the parties are located. We need to know this in order to help us assign the case to the right office. c. asks for the name, address and telephone number of the attorney handling your case. Since you are acting as your own attorney, please indicate that information about you. Section II - Basis of Jurisdiction, asks why you are filing this case in U.S. District ourt. There are only four reasons, as outlined on page one of this booklet. Please indicate the one item that best says why your case is being filed in federal court. Section III - itizenship of Principal Parties, is used only if you indicated Diversity in section II. Pick one box that says where the plaintiff is located and one box to show where the defendant is located. Section IV - Origin, gives 7 options. You will most likely choose option one, original proceeding. That means that you are filing a new lawsuit. The other options are easy to understand. Section V - Nature of Suit, says very generally what your case is about. Please check one box only. Section VI - ause of Action, asks for a brief statement telling us what your case is about. Something like, I am suing my employer for violating my civil rights for not promoting me. You can ignore the part that asks you to cite a civil statute. Section VII is pretty easy to understand. Put down the dollar amount you requested in the complaint, and check if you demanded a jury. Section VIII asks if there are any related cases. If you sued the same defendants in this or any other court, write down the name of the Judge and the case number. Please date and sign the form where it says, Signature of Attorney of Record. The back of the form asks two questions about your case. If you have never sued these defendants before, the answer to both questions would be no. 6
8 About Summonses The summons form is pretty easy to fill out. Just remember that, in the box that asks for the name and address of the defendant being served, you can only put down one name - the party you are suing - and their address. If you are suing more than one person, each individual defendant gets his/her own summons. The lerk s Office cannot issue summonses without this information. Remember, each defendant gets his or her own summons. Do not indicate more than one party and address on a summons form. We should be able to look at your completed summons form and see exactly which part is going to receive it. In the space that gives the defendants the number of days they have to answer, please write in, 20. If you are suing the United States government, enter 60. What Happens After My ase is Filed? You need to let the people you are suing know that you are suing them. You do this by having the deputy clerk sign and seal a summons form. You then serve that form, along with a copy of your complaint, on the defendants. The rules say that you can mail the summons, or it can be served personally by anyone who is not a party and is at least 18 years old. If the filing fee was waived, the ourt may arrange for your defendants to be served. Please remember that when you file documents in your case, you must send or bring an original and a copy of the document to the court. You will also need to serve your defendants. If you are suing the United States Government: The Federal Rules say that when you sue the U.S. government, you will need to serve summonses on three separate parties. They are: The head of the agency you are suing The United States Attorney in Detroit The United States Attorney General in Washington Even if you are suing a local government agency, you need to serve that office s headquarters, usually in Washington, D.. For example, if you were injured at a local Post Office, you would actually serve the Postmaster General in Washington, not the local Post Office. Your summonses should be directed to: Office of the Attorney General Department of Justice 10 th & Pennsylvania Ave., N.W. Washington, D United States Attorney 211 W. Fort Street Suite 2300 Detroit, MI The head of the federal agency that you are suing. Some frequently named defendants include: ommissioner of Social Security Office of the General ounsel 6401 Security Boulevard, Room 611 Baltimore, MD U.S. Postmaster General 475 L Enfant Plaza, S. W. Washington, D Secretary, Department of Treasury 1500 Pennsylvania Ave., N.W. Washington, D If you need an address not shown here, you can get help from the Federal Information enter, (800)
9 JS 44 11/99 IVIL OVER SHEET OUNTY IN WHIH THIS ATION AROSE: The JS-44 civil cover sheet and the information contained herein neither replace nor supplement the filing and service of pleadings or other papers as required by law, except as provided by local rules of court. This form, approved by the Judicial onference of the United States in September 1974, is required for use of the lerk of ourt for the purpose of initiating the civil docket sheet. I. (a) PLAINTIFFS DEFENDANTS (b) ounty of Residence of First Listed () Attorney's (Firm Name, Address, and Telephone Number) ounty of Residence of First Listed NOTE: IN LAND ONDEMNATION ASES, USE THE LOATION OF THE LAND INVOLVED. Attorneys (If Known) 11. BASIS OF JURISDITION (Place an ''X'' in One Box Only) III. ITIZENSHIP OF PRINIPAL PARTIES (Place an ''X'' in One Box for Plaintiff (For Diversity ases Only) and One Box for Defendant) PLA DEF PLA DEF 1 U.S. Government 3 Federal Question itizen of This State 1 1 Incorporated or Principal Place 4 4 Plaintiff (U.S. Government t a Party) of Business In This State 2 U.S. Government 4 Diversity itizen of Another 2 2 Incorporated and Principal 5 5 Defendant (Indicate itizenship of Parties of Business In Another State in Item 111) itizen or Subject of a Foreiqn ountrv 3 3 Foreign Nation 6 6 IV. NATURE OF SUIT (Place an ''X'' in One Box Only) ONTRAT TORTS FORFEITURE/PENALTY BANKRUPTY OTHER STATUTES Insurance PERSONAL INJURY PERSONAL INJURY 610 Agriculture 422 Appeal 28 US State Reapportionment 120 Marine 31 0 Airplane 362 Personal Injury- 620 Other Food & Drug 41 0 Antitrust 130 Miller Act 315 Airplane Product Med. Malpractice 625 Drug Related Seizure 423 Withdrawal 430 Banks and Banking 140 Negotiable Instrument Liability 365 Personal InVry - of Property 21: US ommerce/i 150 Recovery of Product Lia ility 630 Liquor Laws 460 Deportation Overpayment and 320 Assault Libel Enforcement And Slander of Judgment 368 Asbestos Personal 640 R.R. & Truck PROPERTY RIGHTS 470 Racketeer Influenced & Injury Product orrupt Organizations 151 Medicare Act 330 Federal Employers' Liability 650 Airline Regs. 820 opyrights 152 Recovery of Defaulted Liability 660 Occupational 810 Selective Service 830 Patent Student Loans 340 Marine PERSONAL PROPERTY Safety/Health 850 Secu rities/om mod ities/ 840 Trademark (Excl. Veterans) 345 Marine Product 370 Other Fraud 690 Other Exchange 153 Recovery of Overpayment Liability 371 Truth in Lending 875 ustomer hallenge of Veteran's Benefits 350 Motor Vehicle 380 Other Personal LABOR SOIAL SEURITY 12 LIS Stockholders' Suits 355 Motor Vehicle Property Damage 891 Agricultural Acts 71 0 Fair Labor Standards 861 H IA (1 395ff) 190 Other ontract Product Liability 385 Property Damage 892 Economic Stabilization Act Act 862 Black Lung (923) 195 ontract Product Liability 360 Other Personal Product Liability 720 Labor/Mgmt. 893 Environmental Matters Injury 863 DlW/DlWW (405(g)) Relations 894 Energy Allocation Act REAL PROPERTY IVIL RIGHTS PRISONER PETITIONS 864 SSID Title XVI 895 Freedom of 730 Labor/Mgmt. Reporting 865 RSI (405(g)) Information Act 210 Land ondemnation 441 Voting 51 0 Motions to Vacate & Disclosure Act FEDERAL TAX SUITS 900 Appeal of Fee 220 Foreclosure 442 Employment Sentence 740 Railway Labor Act Determination Under 230 Rent Lease & Ejectment 443 Housing/ Habeas orpus: Equal Access to Justice 870 Taxes (U.S. Plaintiff 240 Torts to Land Accommodations 530 General 790 Other Labor Litigation or Defendant) 950 onstitutionality of 245 Tort Product Liability 444 Welfare 535 Death Penalty State Statutes 290 All Other Real Property 440 Other ivil Rights 540 Mandamus & Other 791 Empl. Ret. Inc. 871 IRS-Third Party 890 Other Statutory Actions 550 ivil Rights Security Act 26 US Prison ondition (PLAE AN ''X'' IN ONE BOX ONLY) V. ORIGIN Appeal to Transferred from District another district Reinstated 5 (specify) 7 Judge from Original Removed from Remanded from or 6 Multi district Proceeding State ourt Appellate ourt Litigation Magistrate Reopened V1. AUSE OF ATION (ite the U.S. ivil Statute under which you are filing and write brief statement of cause. Do not cite jurisdictional statutes unless diversity.) Vil. REQUESTED IN OMPLAINT: (See Vill. RELATED ASE(S) instructions): IF ANY HEK IF THIS IS A LASS ATION $DEMAND HEK YES only if demanded in complaint: UNDER F.R..P. 23 JURY DEMAND: JUDGE DOKET NUMBER DATE SIGNATURE OF ATTORNEY OF REORD
10 PURSUANT TO LOAL RULE Is this a case that has been previously dismissed? If yes, give the following information: o u rt: ase.: Judge: 2. Other than stated above, are there any pending or previously discontinued or dismissed companion cases in this or any other court, including state court? (ompanion cases are matters in which it appears substantially similar evidence will be offered or the same or related parties are present and the cases arise out of the same transaction or occurrence.) If yes, give the following information: ourt: ase.: Judge: tes :
11 AO 240 (Rev. 9/96) UNITED STATES DISTRIT OURT EASTERN DISTRIT OF MIHIGAN Plaintiff V. APPLIATION TO PROEED WITHOUT PREPAYMENT OF FEES AND AFFIDAVIT Defendant ASE NUMBER: 1, petitioner/plaintiff/movant other declare that I am the (check appropriate box) in the above-entitled proceeding; that in support of my request to proceed without prepayment of fees or costs under 28 US declare that I am unable to pay the costs of these proceedings and that I am entitled to the relief sought in the complaint/petition/motion. In support of this application, I answer the following questions under penalty of perjury: 1. Are you currently incarcerated? (If '','' go to Part 2) If '','' state the place of your incarceration Are you employed at the institution? Do you receive any payment from the institution? Attach a ledger sheet from the institution(s) of your incarceration showing at least the past six months' transactions. 2. Are you currently employed? a. If the answer is '','' state the amount of your take-home salary or wages and pay period and give the name and address of your employer. b. If the answer is '','' state the date of your last employment, the amount of your take-home salary or wages and pay period and the name and address of your last employer. 3. In the past 12 twelve months have you received any money from any of the following sources? a. Business, profession or other self-employment b. Rent payments, interest or dividends. Pensions, annuities or life insurance payments d. Disability or workers compensation payments e. Gifts or inheritances f. Any other sources If the answer to any of the above is '','' describe, on the following page, each source of money and state the amount received and what you expect you will continue to receive.
12 AO 240 (REV.9/96) Application to Proceed Without Prepayment of Fees and Affidavit - Page 2 4. Do you have any cash or checking or savings accounts? If '','' state the total amount. 5. Do you own any real estate, stocks, bonds, securities, other financial instruments, automobiles or any other thing of value? If '','' describe the property and state its value. 6. List the persons who are dependent on you for support, state your relationship to each person and indicate how much you contribute to their support. I declare under penalty of perjury that the above information is true and correct. Date Signature of Applicant NOTIE TO PRISONERS: A Prisoner seeking to proceed without prepayment of fees shall submit an affidavit stating all assets. In addition, a prisoner must attach a statement certified by the appropriate institutional officer showing all receipts, expenditures, and balances during the last six months in your institutional accounts. If you have multiple accounts, perhaps because you have been in multiple institutions, attach one certified statement of each account.
13 United States District ourt Eastern District of Michigan Summons in a ivil Action and Return of Service Form Plaintiff(s) Name ase Number and Judge Assignment (to be supplied by the ourt) Defendant(s) Name VS. Plaintiffs attorney, address and telephone: Name and address of defendant being served: To the defendant This summons is notification that YOU ARE BEING SUED by the above named plaintiff(s). 1. You are required to serve upon the plaintiff's attorney, name and address above, an answer to the complaint within days after receiving this summons, or take other actions that are permitted by the Federal Rules of ivil Procedure. 2. You must file the original and one copy of your answer within the time limits specified above with the lerk of ourt. 3. Failure to answer or take other action permitted by the Federal Rules of ivil Procedure may result in the issuance of a judgment by default against you for the relief demanded in the complaint. David J. Weaver lerk of the ourt By: Deputy lerk Date of issuance INT-0131-MIE-REV. 12/93 06/99 PAGE 1 OF 2
14 RETURN OF SERVIE A copy of the summons and complaint has been served upon the defendant in the manner indicated below: Name of Defendant served: Date of service: Method of Service Personally served at this address: Left copies at the defendant's usual place of abode with (name of person): At this address: Other (please specify): Service fees: Travel $ Service $ Total $ I declare under the penalty of perjury that the information contained in this Return of Service is true. Date Signature of server Server's printed name Server's address PAGE 2 OF 2
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text:
Fred and Finn
Written by Madeline Goodey
Illustrated by Mike Gordon
Part of the Readzone Books Magpies Series
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Madeline Goodey
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