Status epilepticus in mice deficient for succinate semialdehyde dehydrogenase: GABAA receptor–mediated mechanisms

Status Epilepticus in Mice Deficient forSuccinate Semialdehyde Dehydrogenase:GABAA Receptor–Mediated Mechanisms

Ying Wu, MD,1,2 Andrea Buzzi, PhD,1,2 Marina Frantseva, MD, PhD,1,2 Jose Perez L. Velazquez, PhD,1–3

Miguel Cortez, MD,1,2 Chunche Liu, MSc,1,2 Liqing Shen, BSc,1,2 K. Michael Gibson, PhD,4

and O. Carter Snead III, MD1–3

The epilepsy that occurs in SSADH deficiency has a seizure phenotype similar to that occurring in the SSADH�/�

mouse. We examined the expression and function of the GABAA receptor (GABAAR) in SSADH-deficient mice. A selec-tive decrease in binding of [35S]tert-butylbicyclophosphorothionate was observed in SSADH�/� mice at postnatal day 7that was progressive until the third postnatal week of life when, at the nadir of the decreased [35S]tert-butylbicyclophosphorothionate binding, generalized convulsive seizures emerged that rapidly evolved into status epilep-ticus. We also observed a substantial downregulation of the �2 subunit of GABAAR, a reduction in GABAA-mediatedinhibitory postsynaptic potentials, and augmented postsynaptic population spikes recorded from hippocampal slices. TheSSADH�/� mouse model represents a powerful investigative tool for understanding the pathophysiology of the seizuresassociated with human SSADH deficiency. These data raise the possibility that progressive dysfunction of the GABAARmay be involved in the development of seizures in SSDAH-deficient mice. Elucidation of the precise fundamental mech-anisms of the perturbation of the GABAAR-mediated function in SSADH�/� mice could lead to the development ofnovel treatment modalities designed to reduce the neurological morbidity in children with SSADH deficiency.

Ann Neurol 2006;59:42–52

Succinic semialdehyde dehydrogenase (SSADH) defi-ciency, or �-hydroxybutyric (GHB) aciduria, is a rareautosomal recessive disorder that is characterized by adefect in the degradation of GABA. In the absence ofSSADH, both GABA and GHB accumulate. As a re-sult, SSADH deficiency in human is characterized bymarkedly increased levels of both GHB and GABA inbrain, blood, and urine. Clinically, SSADH deficiencymay present with a wide spectrum of neurological dys-function, including language delay, ataxia, hypotonia,and mental retardation. Epilepsy occurs commonly inSSADH deficiency and is characterized by absence,myoclonic, and convulsive seizures, as well as convul-sive status epilepticus.1–3

One of our investigators (K.M.G) generated and char-acterized SSADH-deficient (SSADH�/�) mice thatshow markedly increased levels of both GABA andGHB in urine and homogenates of liver and brain.4 Inaddition to ataxia and poor weight gain,5 SSADH�/�

mice display absence, myoclonic, and convulsive seizuresand convulsive status epilepticus, a seizure phenotypesimilar to that in human SSADH deficiency.3,5,6 More-over, in the mutant animals, the occurrence of general-ized convulsive seizures and status epilepticus is a devel-opmental phenomenon that emerges late in the thirdpostnatal week of life.6

To understand the cellular events that underlie thedevelopmental appearance of generalized tonic-clonicseizures and status epilepticus in the SSADH�/�

mouse, we examined putative mechanisms of hyperex-citability in the SSADH�/� mouse during the criticaldevelopmental window when absence seizures evolveinto generalized convulsive seizures and status epilepti-cus. Because the mutant animals have markedly in-creased levels of GABA, we focused on putativeGABAergic mechanisms in the evolution from absenceto convulsive seizures with a particular focus onGABAA receptor (GABAAR)–mediated function.

From the 1Brain and Behavior Program, 2Division of NeurologyHospital for Sick Children, 3Department of Pediatrics, Faculty ofMedicine, University of Toronto, Toronto, Ontario, Canada; and4Department of Molecular and Medical Genetics, Oregon Health &Science University, Portland, OR.

Received Apr 18, 2005, and in revised form Aug 8. Accepted forpublication Aug 8, 2005.

Published online Oct 20, 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.20686

Address correspondence to Dr Snead, 555 University Avenue, To-ronto, Ontario, Canada M5G 1X8. E-mail: [email protected]

42 © 2005 American Neurological AssociationPublished by Wiley-Liss, Inc., through Wiley Subscription Services

Materials and MethodsSuccinic Semialdehyde Dehydrogenase Null MiceThe SSADH mouse model was generated by standard genetargeting and characterized by Hogema and colleagues.4

Mice were genotyped by two-allele three-primer polymerasechain reaction using tail genomic DNA, as described previ-ously.4 Experiments were conducted comparing the knock-out (SSADH�/�) versus their littermate wild-type(SSADH�/�) mice used as control at age postnatal day 7(P7) to P19, before the onset of generalized convulsive sei-zures.6 Developing SSADH�/� and SSADH�/� mice wereused for the experiments described in the next section at thetimes specified during the first three postnatal weeks of life.

Binding ExperimentsLIGANDS. [35S]Tert-butylbicyclophosphorothionate ([35S]T-BPS; specific activity of 104Ci/mmol), a GABAAR antagonist,[3H]muscimol (specific activity of 20Ci/mmol), a GABAARagonist, and [3H]flunitrazepam (specific activity of 85Ci/mmol), an agonist at the benzodiazepine site of the GABAARwere purchased from Du-Pont, New England Nuclear (Bos-ton, MA).

[35S]TERT-BUTYLBICYCLOPHOSPHOROTHIONATE AUTORA-DIOGRAPHY. For [35S]TBPS autoradiography and all auto-radiographic binding experiments described later, SSADH�/�

and age-matched wild-type mice were killed by decapitation;the brains were removed and immediately immersed in iso-pentane at �35°C. Coronal sections were cut from the ante-rior to posterior boundaries of the cerebral cortex at 20�m at�20°C and thaw-mounted onto gelatin-coated slides thatwere dried and stored at �80°C until used.7 [35S]TBPS bind-ing was performed by a modification of the method of Edgarand Schwartz.8 The regions analyzed were the frontoparietalcortex, the ventrobasal thalamus, and the CA1 region of thehippocampus. The precise location of these regions was as de-scribed previously.7

[35S]TERT-BUTYLBICYCLOPHOSPHOROTHIONATE BINDINGKINETICS. The kinetics of [35S]TBPS binding to synapticmembranes was determined in SSADH�/� and age-matchedwild-type mice as Cross and colleagues9 described previously.Nonspecific binding was determined in the presence of100�M picrotoxin and represented less than 10% of the to-tal binding at concentrations near the apparent dissociationconstant (Kd) for [35S]TBPS binding. Protein quantificationwas determined by BCA assay (Pierce, Rockford, IL).

[3H]MUSCIMOL AUTORADIOGRAPHY. [3H]Muscimol bind-ing was performed on SSADH�/� and age-matched wild-typecontrol mice as Titulaer and colleagues10 described, but withmodifications. In brief, tissue slices were prewashed in 50mMtris(hydroxymethyl)aminomethane (Tris)-citrate buffer (pH7.1), 150mM NaCl, for 30 minutes at 4°C and air-dried. Sec-tions were incubated in 10nM [3H]muscimol in 50mM Tris-citrate buffer for 40 minutes at 4°C, rinsed 3 times in cold50mM Tris-citrate buffer for 5 minutes each, dipped once inice-cold distilled water, and air-dried. Nonspecific binding wasdetermined in the presence of 100�M muscimol and repre-sented less than 10% of the total binding.

[3H]FLUNITRAZEPAM AUTORADIOGRAPHY. [3H]Flunitraz-epam binding was performed on SSADH�/� and age-matched wild-type control mice by a modification of themethod of Carlson and colleagues.11 A total of 10�M fluni-trazepam was used to determine nonspecific binding, whichrepresented less than 10% of the total binding.

ANALYSIS OF BINDING. Five mutant and five wild-typecontrol animals were used in the autoradiographic studies,and each experiment was done in triplicate. The autoradio-graphic data were analyzed as reported elsewhere.12 Dried tis-sue sections were opposed to hyperfilm-bmax film (Amer-sham, Arlington, IL) with [3H] microscale standards(Amersham) for 2 to 3 weeks at room temperature. Thefilms were developed in D-19 (Kodak, Rochester, NY), fixed,and air-dried. Quantitative analysis of the resulting auto-radiograms was performed densitometrically using amicrocomputer-based densitometer system (MCID; ImagingResearch; Ontario, Canada). In brief, a standard curve be-tween the optical density of [3H] standards and tissue radio-activity equivalents (pmol/mg of tissue) was constructed us-ing a nonlinear regression analysis. The average opticaldensity values of the selected brain regions were in the linearportion of this standard curve. The value (measured in pmol/mg) in each brain region was calculated by interpolation us-ing the image analyzer.13 Five to eight readings were deter-mined and averaged for each anatomic area analyzed.

Immunoblotting and ImmunohistochemistryIMMUNOBLOTTING. SSADH�/� and age-matched SSAD-H�/� mice were killed by decapitation under light halothaneanesthesia; brains were immediately excised, and cerebral cor-tex, hippocampus, and thalamus were dissected on ice-coldglass. The anatomic dissections were performed as describedpreviously.14,15 A variety of primary antibodies were used toprobe the membranes. For GABAAR, anti-GABAA �1 (LabVision, Fremont, CA), anti-GABAA �2 (1:500; Novus Bio-logicals, Littleton, CO), and anti-GABAA �2 (1:1,000;Chemicon International, Temecula, CA) subunits (2-hourincubation at room temperature), rabbit polyclonal antibod-ies were used. Appropriate rabbit (Vector Laboratories, Bur-lingame, CA) secondary horseradish conjugated antisera wereused in the secondary incubation (1 hour at room tempera-ture), and then developed by enzyme chemiluminescence(Amersham Pharmacia). The antibody is specific against bothGABAA �2 heavy (H) and light chain (L). There is noknown reactivity with other GABA subunits according to themanufacturer.

IMMUNOHISTOCHEMISTRY. Brains were excised andchilled in isopentane (�40°C) for 1 minute and brought to�20°C for cryosectioning. Sections were fixed in ice-coldphosphate-buffered saline (PBS), 4% paraformaldehyde for10 minutes, then rinsed in PBS and immersed for 5 minutesat room temperature in 0.3% H2O2 prepared in PBS toblock endogenous peroxidase. After a fast wash, sections werepreincubated for 2 hours in 5% normal goat serum PBS with0.25% Triton-X100 (Sigma Labs, St. Louis, MO) to blocknonspecific reactions, and then incubated in rabbit anti-GABAA �1 (1:200; Lab Vision) or �2 antiserum (1:300;

Wu et al: Receptors and Seizures 43

Novus Biologicals) overnight at 4°C with shaking. Secondaryincubation, with horseradish peroxidase–conjugated goatanti–rabbit IgG (1:500; Vector Laboratories), was performedat room temperature for 1 hour. The immune complexeswere visualized with enhanced metal diaminobenzidine tetra-hydrochloride (Pierce).

Electrophysiological ExperimentsBRAIN SLICES AND SOLUTIONS. P8- to P14-old mice (8SSADH�/� and 10 SSADH�/� mice) were anesthetizedwith halothane and decapitated to obtain hippocampal slices.Transverse brain slices (450�m) were obtained by a Vi-bratome (Series 1000; Technical Products International (El-lisville, MD), St. Louis, MO) and maintained in artificialcerebrospinal fluid containing 125mM NaCl, 2.5mM KCl,1.25mM NaH2PO4, 2mM MgSO4, 2mM CaCl2, 25mMNaHCO3, and 10mM glucose. To investigate possible epi-leptiform activity (specifically primary or secondary afterdis-charges, or both), we increased KCl concentration to 5mMand decreased the concentration of MgSO4 to 0.9mM.When needed, drugs were applied by superfusion. D-2-amino-5-phosphopentanoic acid (D-AP5; 20�M, made of50mM stock solution in distilled water) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100�M, made of50mM stock in dimethylsulfoxide) were obtained from Toc-ris Cookson and diluted daily.

Electrophysiological RecordingsWHOLE-CELL RECORDINGS. Neuronal recordings wereobtained with the use of the whole-cell configuration of thepatch-clamp technique16 from the CA1-CA2 hippocampalpyramidal neurons. Electrodes were filled with internal solu-tion of 150mM potassium gluconate, 10mM N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid (HEPES),2mM Mg-ATP, 5mM KCl, and 0.1mM EGTA; osmolaritywas 275 � 5mOsm, pH 7.2 adjusted with potassium hy-droxide. Electrodes had tip resistances between 5 and 6 M.Neuronal responses were recorded with the use of an Axoc-lamp 2-A amplifier (Axon Instruments, Foster City, CA) inbridge mode. For extracellular stimulation, a bipolar stimu-lation electrode was placed in the Schaffer collaterals. Initiallow-stimulus intensities were gradually increased to elicit sta-ble postsynaptic responses (as measured by either extracellu-lar local field potentials or whole-cell recorded potentials forat least 15–20 minutes) and were maintained at this level forthe duration of all the experiments that required this type ofstimulation.

EXTRACELLULAR RECORDINGS. For extracellular stimula-tion, current pulses (single-current stimuli 150 millisecondsat 0.66Hz) were delivered through a bipolar stimulating elec-trode positioned in the Schaffer collaterals. The extracellularrecording electrode was filled with artificial cerebrospinalfluid and placed in the CA1 cell body layer. Electrical signalswere recorded using an Axoclamp 2A amplifier (Axon Instru-ments). PCLAMP software (Axon Instruments) was used foranalysis of membrane potential, input resistance (which wasmeasured from the linear part of the current-voltage plot),and amplitude of the postsynaptic responses. The amplitudes

of the postsynaptic responses were measured from the peakto the baseline.

Statistical AnalysisStatistical analysis of [35S]TBPS binding at different ages ofSSADH�/� mice were analyzed by one-way analysis of vari-ance followed by Newman–Keuls multiple-comparisons test.Two individual group means were compared using a two-tailed, independent Student’s t test. Quantification of immu-noblotting was obtained by dosimeter. Each lane was loadedwith 15�l of samples, and the loading amount normalizationwas obtained by comparison with the antiactin band on thesame blot. Two individual group means were compared us-ing a two-tailed, independent Student’s t test. The unpairedStudent’s t test was used in all of electrophysiological exper-iments. Significance was considered at p 0.05; numericalvalues are expressed as means � standard error.

ResultsBinding Experiments[35S]TERT-BUTYLBICYCLOPHOSPHOROTHIONATE BINDING

IN SUCCINIC SEMIALDEHYDE DEHYDROGENASE NULL MICE

SHOWS A PROGRESSIVE DECREASE WITH DEVELOPMENT.

The seizure phenotype of the SSADH�/� mouse hasbeen described.6 In brief, spontaneous, recurrent,absence-like seizures appeared during the second weekof life at P14. These seizures meet all of the behavioral,electrophysiological, and electrographic criteria for ab-sence seizues.17 The absence seizures persisted untilaround P18 to P20, when they were superseded by theoccurrence of myoclonic seizures that evolved rapidlyinto generalized convulsive seizures. Status epilepticusthen rapidly emerged and often was lethal by P23.Three specific radiolabeled ligands, [35S]TBPS,[3H]muscimol, and [3H]flunitrazepam, were first usedto assess the binding properties of GABAAR in fresh-frozen brain sections at P7, before the onset of absenceseizures, and from P14 to P19, during the evolution ofabsence to generalized convulsive seizures. We foundthat [35S]TBPS binding in SSADH�/� mice was signif-icantly reduced at P7 and P14 when compared withwild-type mice and became progressively more dimin-ished until the third postnatal week of life just before theonset of generalized convulsive seizures (Fig 1). A signif-icant (p 0.01; n � 5) reduction in [35S]TBPS bind-ing was observed in SSADH�/� mice before the devel-opmental onset of generalized convulsive seizures (seeFigs 1A, B). The decrease in [35S]TBPS was observedthroughout all brain regions examined (see Fig 1C) andwas progressive over the first 3 weeks of life (see Fig1D). One-way analysis of variance showed there was asignificant difference of [35S]TBPS binding when miceof different ages were compared, with the older micehaving significantly lower [35S]TBPS binding. P14showed significantly lower [35S]TBPS binding than P7(p 0.001; n � 5), and P19 showed significantly lower

44 Annals of Neurology Vol 59 No 1 January 2006

binding than P14 (p 0.001; n � 5). Linear regressionanalysis also showed that the decrease in [35S]TBPSbinding in SSADH�/� mice was significantly age de-pendent in all three regions measured (p 0.001; n �5). Scatchard analysis of [35S]TBPS binding at P18, be-fore onset of seizures, showed a significant (p 0.01;n � 3) decrease in Bmax in SSADH�/� (720.3 �127.2fmol/mg) versus SSADH�/� mice (2,051.0 �170.5fmol/mg). There was no significant difference be-tween SSADH�/� and SSADH�/� mice in Kd of

[35S]TBPS binding. SSADH�/� mice showed a Kd of3.87 � 0.60 versus 3.40 � 1.17nM in the wild-typecontrol animals (see Fig 1E).

SUCCINIC SEMIALDEHYDE DEHYDROGENASE NULL MICE

SHOWED NO CHANGES IN OTHER GABAA RECEPTOR LI-

GANDS. [3H]Muscimol and [3H]flunitrazepam bind-ing studies provided no evidence for a significant dif-ference between SSADH�/� and SSADH�/� mice

Fig 1. [35S]Tert-butylbicyclophosphorothionate ([35S]TBPS) autoradiography in succinic semialdehyde dehydrogenase null(SSADH�/�) mice. Representative autoradiography demonstrated a decrease of [35S]TBPS binding in the brain sections of postnatalday 19 (P19) SSADH�/� mice before the onset of tonic-clonic seizures (B) when compared with that of P19 SSADH�/� mice(A). Quantitative analysis (C) showed that [35S]TBPS binding was significantly reduced in cortex, hippocampus, and thalamus(arrows) of SSADH�/� mice. Values represent percentages (means � standard error; n � 5) of [35S]TBPS binding in SSADH�/�

mice. Student’s unpaired t test was used to compare with control value (*p 0.05; **p 0.01). The analysis of linear regressionshowed that the decrease in [35S]TBPS was significantly dependent on the age of the SSADH�/� mouse in all three regions mea-sured (p 0.001; n � 5) (D). Scatchard analysis (E) showed a significant decrease of [35S]TBPS binding in Bmax in SSADH�/�

when compared with SSADH�/� mice: Bmax in SSADH�/� (720.3 � 127.2fmol/mg) versus SSADH�/� (2,051.0 � 170.5fmol/mg). Three independent experiments were done with triplicates in each experiment (means � standard error; n � 5; done in trip-licate). Student’s unpaired t test was used to compare with control value (p 0.01).


during the developmental time period under examina-tion (Figs 2A–D).

Immunoblotting and Immunohistochemistry ResultsSUCCINIC SEMIALDEHYDE DEHYDROGENASE NULL MICE

SHOWED DECREASED EXPRESSION OF GABAA RECEPTOR

�2, BUT NOT GABAA RECEPTOR �1, GABAA RECEPTOR �2.

[35S]TBPS binding is GABAAR subunit dependent.18,19

Therefore, the significant decrease in [35S]TBPS bindingand decrease in Bmax for [35S]TBPS binding observed inSSADH�/� mice early in life raised the possibility ofaltered subunit expression of GABAAR. Immunohisto-chemical staining showed no difference betweenSSADH�/� and SSADH�/� mice in the expressionpattern of GABAA �2, a pattern of expression that wassimilar to that reported previously in normal rat brain.20

However, the immunohistochemical data suggest thatthere was a decrease in GABAA �2 expression inSSADH�/� mice that was present throughout all brainareas (Fig 3A). Quantitative immunoblotting analysis

showed a significant decrease of GABAA �2 subunits(see Figs 3C, D), but not GABAAR �1, GABAAR �2

(Figs 3B, C), and GABAA �3 subunits (data not shown),in the neocortex, hippocampus, and cerebellum ofSSADH�/� mice. The decrease of GABAA �2 in thethalamus of SSADH�/� mice did not reach significance(data not shown). The magnitude of the observed de-crease in GABAA �2 in SSADH�/� mice did not appearto be as great as that observed for [35S]TBPS binding inthe mutant animals.

Electrophysiological ExperimentsHIPPOCAMPAL SLICES FROM SUCCINIC SEMIALDEHYDE

DEHYDROGENASE NULL MICE ARE HYPEREXCITABLE.

Electrophysiological recordings from wild-type andSSADH�/� hippocampal slices were used to examinethe epileptiform activity observed in SSADH�/� mice.There were two groups of animals: at P7 to P8 and atP14 to P16. Hyperexcitability was observed with extra-cellular recordings only in the P14 to P16 group. Extra-

Fig 2. [3H]Flunitrazepam and [3H]muscimol autoradiography in succinic semialdehyde dehydrogenase null (SSADH�/�) mice. Rep-resentative autoradiograph of [3H]flunitrazepam (A, B) and [3H]muscimol binding (C, D) in brain sections of SSADH�/� (A,C)and SSADH�/� (B, D) mice at postnatal day 19 (P19), before the onset of generalized tonic clonic seizures, showed no significantdifferences.


cellular recordings of somatic field potentials in CA1 py-ramidal layers showed spontaneous local field potentialssynchronous events in 4 of 6 (67%) hippocampal slicesfrom SSADH�/� (n � 6 mice) and 0 of 6 slices fromSSADH�/� mice at P13 to P14 days of age (Fig 4A). Inaddition, primary afterdischarges were observed in re-sponse to 15 to 100Hz train in 5 of 6 (83%) hippocam-pal slices from SSADH�/� mice (n � 7 mice), with amean afterdischarge duration of 16.1 � 5.5 seconds, butno afterdischarges were found in wild-type mice hip-pocampal slices (0/6). Input-output curves, calculatedfrom the evoked postsynaptic responses of CA1 pyrami-dal layers (the amplitudes of the peak evoked responseswere measured as detailed earlier in Materials and Meth-ods), further illustrate the increased excitability in the

slices from knockout animals (see Figs 4B, C). The abil-ity of the mutant neurons to respond to electrical stim-ulation of Schaffer collaterals was significantly greater inSSADH�/� mice, as judged by the higher amplitudesand greater number of population spikes evoked by asingle pulse stimulation to the Schaffer collaterals re-corded in the SSADH�/� slices. These data indicatethat hippocampal slices from SSADH�/� mice are hy-perexcitable in the second week of life.

SUCCINIC SEMIALDEHYDE DEHYDROGENASE NULL MICE

SHOW ALTERATIONS IN INTRINSIC MEMBRANE PROPER-

TIES AND GABAA RECEPTOR–MEDIATED INHIBITORY NEU-

ROTRANSMISSION. Passive membrane properties andinhibitory neurotransmission were estimated using

Fig 3. GABAA receptor (GABAAR) subunit expression in succinic semialdehyde dehydrogenase null (SSADH�/�) mice. Immunohis-tochemical staining showed a universal downregulation of GABAAR �2 in the brain of SSADH�/� mice when compared with age-matched SSADH�/� mice (A). However, quantitative immunoblotting analysis (C) only confirmed the decrease of GABAAR �2

heavy (H) and light chain (L) as being significant in neocortex (CTX), hippocampus (HIP), and cerebellum (CEB) of SSADH�/�

mice when compared with SSADH�/� mice at postnatal day 17 (P17) to P19 (D). Fifteen-microliter protein from brain wereresolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose as described. Values representpercentages (means � standard deviation; n � 3) of the band intensity of GABAAR �2 in SSADH�/� mice. Student’s unpaired ttest showed that significant decrease of GABAAR �2 in SSADH�/� in comparison with control value (*p 0.05; **p 0.01).There was no significant change of expression of GABAAR �1 and GABAAR �2 (B, C).


whole-cell patch-clamp recordings of pyramidal hip-pocampal neurons in SSADH�/� and wild-type con-trol mice. As shown in Figure 5, there was no differ-ence in input resistance and resting membranepotential recorded at P8 (see Fig 5A); however, by P14,a significant reduction in input resistance and depolar-

ization of resting membrane potential had emerged inSSADH�/� mice (see Fig 5B).

Inhibitory neurotransmission was estimated from in-tracellular postsynaptic responses in the presence of theglutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphopentanoic acid (to

Fig 4. Extracellular field recordings in hippocampal CA1 pyramidal layers demonstrate hyperexcitability in succinic semialdehydedehydrogenase null (SSADH�/�) mice after postnatal day 14 (P14). (A) Spontaneous rhythmic activity was recorded in 67% ofSSADH�/� brain slices (top trace), whereas no spontaneous discharges were obtained from SSADH�/� slices (bottom trace). Ampli-tude of evoked postsynaptic field potentials (B) and the number of population spikes (C) are larger in mutant mice (n � 6SSADH�/�; n � 5 SSADH�/�). Somatic field potentials were recorded from CA1 pyramidal layers, the stimulating electrode wasplaced in the Schaffer fibers, and the stimulation intensity was in log scale (x-axis). (D) Two local field potentials recordings in theCA1 cell body layer. Arrows indicate the stimulating artifact.


block excitatory postsynaptic potentials). Extracellularstimulation of the Schaffer collaterals evoked GABAA-mediated inhibitory postsynaptic potentials that wereblocked by bicuculline. GABAA responses from the neu-rons of SSADH�/� mice were significantly lower thaninhibitory responses elicited from control mice at P8 andP14 (Figs 6A, B).

DiscussionThese data show a significant and progressive selectivedecrease in [35S]TBPS binding in SSADH�/� micethat is age dependent. The decrease in [35S]TBPSbinding progressed from P7 to P19 where a nadir wasreached that was coincident with the onset of general-ized convulsive seizures in the SSADH�/� mice inwhich spontaneous, recurrent, generalized absence sei-

zures emerged reliably at P14.6 The decrease in [35S]T-BPS binding was associated with a smaller decrease inthe expression of GABABR �2, as well as decreasedGABAAR-mediated inhibitory postsynaptic potentialsand increased excitability in hippocampal of the mu-tant mice. These data are significant, but only correla-tive, because they do not address directly the issue of acause–effect relation between the decrease in GABAAR-mediated transmission during the first 3 weeks of lifein the SSADH�/� mice and the progressive evolutionof absence seizures into severe and ultimately fatal gen-eralized convulsive seizures.

The mechanism by which absence seizures in juve-nile absence epilepsy transition to generalized convul-sive seizures in children is completely unknown, be-cause until now, with the advent of the SSADH�/�

Fig 5. Comparison of intrinsic membrane properties in hippocampal neurons of succinic semialdehyde dehydrogenase null(SSADH�/�) and SSADH�/� mice at postnatal day 8 (P8) and P14. Although there was no difference in input resistance andresting membrane potential recorded from P8 animals (A), a significant reduction in input resistance and resting membrane poten-tial was observed in SSADH�/� mice after P14 (B). Whole-cell recordings show the voltage deflections, in two pyramidal cells, forthe same hyperpolarizing current pulses (C). These responses were used to estimate the input resistances of the cells.


mouse model,6 there has been no animal model of gen-eralized absence seizures that reflect this transition. Inthis regard, the SSADH�/� mouse model is quite dif-ferent from pharmacological and genetic models of ab-sence seizures, where absence seizures do not progressto generalized convulsive seizures.17 The SSADH�/�

mouse model differs also in the involvement of struc-tures beyond the thalamocortical circuitry, because theCA1 region of the hippocampus, a region not involvedin rodent models of typical absence seizures,17 showeddecreased GABAAR-mediated function in these experi-ments. In fact, the involvement of limbic, as well asthalamocortical, circuitry in SSADH�/� mice mayhelp to explain why seizure course in these animalsprogresses to generalized convulsive seizures.

A unitary hypothesis that would take into accountthe currently available data to explain the absence andgeneralized convulsive seizures in the SSADH�/�

mouse is that the absence seizures in the SSADH�/�

mouse that appear in the second week of life resultfrom markedly increased levels of GHB in the brains ofthe mutant animals. GHB is well known to induce thiskind of seizure in experimental animals,21 and the de-velopmental profile fits that of GHB-induced absence

seizures.22 However, GHB does not induce generalizedconvulsive seizures in rodents21; therefore, the general-ized convulsive seizures and status epilepticus in theSSADH�/� mouse could arise from decreasedGABAAR-mediated inhibition induced by use-dependent downregulation of GABA receptors second-ary to the markedly increased levels of GABA in thebrains of SSADH�/� mice. The resultant progressivedecrease in GABAAR-mediated inhibition in SSADHbrain would initially be heralded by an increase in ex-citability, as shown in the hippocampal slice data, andwould culminate in the onset of generalized convulsiveseizures23 later in postnatal life.

Prolonged occupancy of GABAARs by ligands, in-cluding GABA, sets in motion a series of mechanismsthat can be termed use-dependent regulation.24 TheSSADH�/� mice have inordinately increased levels ofGABA that could lead to prolonged occupancy ofGABAAR, and hence use-dependent downregulation.Downregulation of GABAAR has been reported in cellculture over a period of hours,25 days,26–28 andweeks,29 as well as in response to systemic administra-tion of progesterone,30 a neurosteroid allosteric modu-lator of GABAAR, and alcohol,31,32 a well-known

Fig 6. GABAA receptor (GABAAR)–mediated inhibitory postsynaptic potentials (IPSPs) are significantly reduced in hippocampalneurons of succinic semialdehyde dehydrogenase null (SSADH�/�) mice at postnatal day 8 (P8) (A) and P14 (B). Amplitudes weremeasured from the intracellular postsynaptic responses in reaction to extracellular stimulus (Schaffer collaterals) in the presence ofd-2-amino-5-phosphopentanoic acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Note that resting membrane po-tential was held at �50mV for P8 and at �60mV for P14 animals. (C) Typical inhibitory potentials (IPSPs) recorded in pyrami-dal cells after stimulation of the stratum radiatum; the left trace shows both GABAA and GABAB components, and the right traceshows the GABAA potential that remains after blockade of GABAB receptors. The recordings correspond to a cell of SSADH�/�

mouse (P14). Amplitudes of the GABAA potentials were measured from the negative peak of the response, as described in Materialsand Methods.


GABAAR receptor agonist.33 Chronically increasedGABA levels also can downregulate phasic GABA re-lease and reduce presynaptic signaling via GABABR,another putative mechanism in the SSADH�/�

mouse.34

Binding to the GABAAR ion channel site complex issubunit dependent.19 TBPS is more specific for thechannel itself35,36 than is muscimol or flunitrazepam.Therefore, in view of the observed dynamic changes inTBPS binding, our data may reflect a gradual develop-mental reduction in GABAAR-gated Cl� function inthe SSADH�/� mouse. There is also ample precedentfor an isolated change in TBPS binding with alter-ations of �2/3,37,38 and � subunits of the GABAAR ap-pear to be involved in [35S]TBPS binding.18,38–41 Iso-lated changes in the steroid modulation of TBPSbinding also have been demonstrated to be associatedwith GHB-induced absence seizures.42 Conversely, it ispossible to get selected alterations in muscimol andflunitrazepam binding with no alterations in TBPSbinding with other pharmacological manipulations.43

These [35S]TBPS binding data raised the possibilityof a corresponding decrease in expression of � subunitsof the GABAAR in SSADH�/� brains, and that iswhat was observed. Indeed, the significant decrease of�2 GABAAR subunit expression in SSADH�/� micesuggests that the progressive decrease in [35S]TBPSbinding in SSADH�/� mice observed from early indevelopment until the onset of generalized convulsiveseizures in the third postnatal week of life may be aconsequence of decreased GABAAR �2 protein expres-sion. The absence of a change in GABAAR �2 wouldsuggest that in this mutant animal, the selective de-crease of �2 may have triggered the dysfunction ofGABAAR-mediated inhibition, as demonstrated elec-trophysiologically. However, the discrepancy betweenthe magnitude of the decrease of TBPS binding andthat of the downregulation of GABAAR �2 suggeststhat other subunit(s) of GABAAR also may be involvedin the decrease of TBPS binding; therefore, thoroughscreening of the expression of all subunits of GABAARin SSADH�/� mice may be helpful in understandingthe mechanism of the decrease of TBPS binding in themutant animal with development.

It is interesting to compare the phenotype of theSSADH�/� mouse with the perturbations ofGABAAR-mediated inhibition reported herein withthat of the GABAAR �3 null mouse. Whereas the latteris characterized by absence-like seizures and hyperactiv-ity,44 the developmental progression from absence sei-zures to lethal status epilepticus6 that occurs in theSSADH�/� mouse is not observed in the �3 nullmouse, suggesting a more generalized deficit ofGABAAR-mediated inhibition in SSADH�/�.

At a cellular level, GABAAR function is controlledby receptor synthesis, assembly, clustering, and cell-

surface expression.45 The difference between GABAARprotein expression and GABAAR binding observed inthe SSADH�/� mouse raises the possibility that themechanism for the observed decreased GABAAR-mediated activity in SSADH and resultant seizurescould be posttranslational. One potential posttransla-tional mechanism in this regard is decreased cell-surface expression of GABAAR in SSADH�/� due toincreased receptor endocytosis in the presence of in-creased ambient levels of GABA in mutant mousebrain. A number of studies support the idea thatpostsynaptic GABAARs cycle between synaptic sitesand intracellular compartments,46 and that het-erodimerization protein–protein interactions betweenGABAAR and other nonionotropic receptors can influ-ence the endocytosis of GABAAR with a resultant pro-found influence on GABAAR-mediated inhibition.47

Thus, any mechanism that regulates the rate of endo-cytosis of GABAAR is predicted to have profound ef-fects on neuronal excitability48 and could well play arole in epileptogenesis in the SSADH�/� mouse.

This work was supported by the NIH (National Disorders of Neu-rological Disorders and Stroke, NS 40270, O.C.S., K.M.G.), Cana-dian Institutes of Health Research (O.C.S.), and members of thePartnership for Pediatric Epilepsy Research (including the AmericanEpilepsy Society, the Epilepsy Foundation, Anna and Jim Fantaci,Fight Against Childhood Epilepsy and Seizures, Neurotherapy Ven-tures Charitable Research Fund, and Parents Against Childhood Ep-ilepsy, O.C.S.).

References1. Gibson KM, Hoffmann GF, Hodson AK, et al.

4-Hydroxybutyric acid and the clinical phenotype of succinicsemialdehyde dehydrogenase deficiency, an inborn error ofGABA metabolism. Neuropediatrics 1998;29:14–22.

2. Pearl PL, Gibson KM, Acosta MT, et al. Clinical spectrumof succinic semialdehyde deficiency. Neurology 2003;60:1413–1417.

3. Dervent A, Gibson KM, Pearl PL, et al. Photosensitive absenceepilepsy with myoclonias and heterozygosity for succinic semi-aldehyde dehydrogenase (SSADH) deficiency. Clin Neuro-physiol 2004;1153:1417–1422.

4. Hogema BM, Gupta M, Senephansiri H, et al. Pharmacologicrescue of lethal seizures in mice deficient in succinate semialde-hyde dehydrogenase. Nat Genet 2001;29:212–216.

5. Gupta M, Hogema BM, Grompe M, et al. Murine succinatesemialdehyde dehydrogenase deficiency. Ann Neurol 2003;54(suppl 6):S81–S90.

6. Cortez MA, Wu Y, Gibson KM, Snead OC. Absence seizuresin succinic semialdehyde dehydrogenase deficient mice: a modelof juvenile absence epilepsy. Pharmacol Biochem Behav 2004;79:547–553.

7. Snead OC, Hechler V, Vergnes M, Maitre M. Increased�-hydroxybutyric acid receptors in thalamus of a genetic animalmodel of petit mal epilepsy. Epilepsy Res 1990;7:121–128.

8. Edgar PP, Schwartz RD. Localization and characterization of35S-t-butylbicyclophosphorothionate binding in rat brain: anautoradiographic study. J Neurosci 1990;10:603–612.


9. Cross AJ, Stirling JM, Robinson TN, et al. The modulation bychlormethiazole of the GABAA-receptor complex in rat brain.Br J Pharmacol 1989;98:284–290.

10. Titulaer MN, Kamphuis W, Pool CW, et al. Kindling inducestime-dependent and regional specific changes in the [3H]mus-cimol binding in the rat hippocampus: a quantitative autora-diographic study. Neuroscience 1994;59:817–826.

11. Carlson BX, Mans AM, Hawkins RA, Baghdoyan HA.Pentobarbital-enhanced [3H]flunitrazepam binding throughoutthe rat brain: an autoradiographic study. J Pharmacol Exp Ther1992;263:1401–1414.

12. Snead OC. Evidence for a G protein-coupled gamma-hydroxybutyric acid receptor. J Neurochem 2000;75:1986–1996.

13. Banerjee PK, Hirsch E, Snead OC. Gamma-hydroxybutyricacid induced spike and wave discharges in rats: relation to high-affinity [3H]gamma-hydroxybutyric acid binding sites in thethalamus and cortex. Neuroscience 1993;56:11–21.

14. Anderson RA, Ritzmann RF, Tabakoff B. Formation ofgamma-hydroxybutyrate in brain. J Neurochem 1977;28:633–639.

15. Snead OC, Morley BJ. Ontogeny of �-hydroxybutyric acid I:regional concentration in developing rat, monkey and humanbrain. Brain Res 1981;227:579–589.

16. Hamill OP, Marty A, Never E, et al. Improved patch-clamptechniques for high resolution current recording from cells andcell-free membrane patches. Pflugers Arch 1981;391:85–100.

17. Snead OC, Depaulis A, Vergnes M, Marescaux C. Absenceepilepsy: advances in experimental animal models. Adv Neurol1999;79:253–278.

18. Slany A, Zezula J, Tretter V, Sieghart W. Rat beta 3 subunitsexpressed in human embryonic kidney 293 cells form high af-finity [35S]t butylbicyclophosphorothionate binding sites mod-ulated by several allosteric ligands of gamma-aminobutyric acidtype A receptors. Mol Pharmacol 1995;48:385–391.

19. Rudolph U, Mohler H. Analysis of GABAA receptor functionand dissection of the pharmacology of benzodiazepines andgeneral anesthetics through mouse genetics. Annu Rev Pharma-col Toxicol 2004;44:475–498.

20. Benke D, Fritschy JM, Trzeciak A, et al. Distribution, preva-lence, and drug binding profile of gamma-aminobutyric acidtype A receptor subtypes differing in the beta-subunit variant.J Biol Chem 1994;269:27100–27107.

21. Snead OC. �-Hydroxybutyric and absence seizures activity. In:Tunnicliff G, Cash CD, eds. Gamma-hydroxybutyrate: molec-ular, functional and clinical aspects. London: Taylor & Francis,2002:132–149.

22. Snead OC. Ontogeny of gamma-hydroxybutyric acid II: elec-troencephalographic effects. Brain Res 1984;317:89–96.

23. Treiman DM. GABAergic mechanisms in epilepsy. Epilepsia2001;42(suppl 3):8–12.

24. Barnes EM. Use-dependent regulation of GABAA receptors. IntRev Neurobiol 1996;39:53–76.

25. Casasola C, Bargas J, Arias-Montano J-A, et al. Hippocampalhyperexcitability induced by GABA withdrawal is due to down-regulation of GABAA receptors. Epilepsy Res 2001;47:257–271.

26. Maloteaux JM, Octave JN, Gossuin A, et al. GABA inducesdown-regulation of the benzodiazepine-GABA receptor com-plex in the rat cultured neurons. Eur J Pharmacol 1987;144:173–183.

27. Baumgartner BJ, Harvey RJ, Darlison MG, Barnes EM. Devel-opmental up-regulation and agonist-dependent down-regulationof GABAA receptor subunit mRNAs in chick cortical neurons.Mol Brain Res 1994;26:9–17.

28. Valeyev AY, Hackman JC, Holohean AM, et al. GABA-induced Cl- current in cultured embryonic human dorsal rootganglion neurons. J Neurophysiol 1999;82:1–9.

29. Redecker C, Luhmann HJ, Hagemann G, et al. Differentialdownregulation of GABAA receptor subunits in widespreadbrain regions in the freeze-lesion model of focal cortical malfor-mations. J Neurosci 2000;20:5045–5053.

30. Czlonkowska AI, Krzascik P, Sienkiewicz-Jaros H, et al. Rapiddown-regulation of GABA-A receptors after pretreatment ofmice with progesterone. Pol J Pharmacol 2001;53:385–388.

31. Petrie J, Sapp DW, Tyndale RF, et al. Altered GABA(A) recep-tor subunit and splice variant expression in rats treated withchronic intermittent ethanol. Alcohol Clin Exp Res 2001;25:819–828.

32. Daglish MR, Nutt DJ. Brain imaging studies in human addicts.Eur Neuropsychopharmacol 2003;13:453–458.

33. Davies M. The role of GABAA receptors in mediating the ef-fects of alcohol in the central nervous system. J Psychiatr Neu-rosci 2003;28:263–274.

34. Jensen K, Chiu C-S, Sokolova I, et al. GABA transporter-1(GABAB1)-deficient mice: differential tonic activation ofGABAA versus GABAB receptors in the hippocampus. J Neu-rophysiol 2003;90:2690–2701.

35. Behrends JC. Modulation by bicuculline and penicillin ofthe block by t-butyl-bicyclo-phosphorothionate (TBPS) ofGABA(A)-receptor mediated Cl(-)-current responses in rat stri-atal neurones. Br J Pharmacol 2000;129:402–408.

36. Fisher JL, Macdonald RL. Functional properties of recombi-nant GABA(A) receptors composed of single or multiple betasubunit subtypes. Neuropharmacology 1997;36:1601–1610.

37. Reynolds DS, Rosahl TW, Cirone J, et al. Sedation and anes-thesia mediated by distinct GABA(A) receptor isoforms. J Neu-rosci 2003;23:8608–8617.

38. Jursky F, Fuchs K, Buhr A, et al. Identification of amino acidresidues of GABAA receptor subunits contributing to the for-mation and affinity of the ter-butylbiciclophosphorothionatebinding site. J Neurochem 2000;74:1310–1316.

39. Luddens H, Seeburg PHS, Korpi ER. Impact of beta andgamma variants on ligand binding properties of gamma-aminobutyric acid type A receptors. Mol Pharmacol 1994;45:810–814.

40. Korpi ER, Kuner T, Seeburg PH, Luddens H. A selective an-tagonist for the cerebellar granule cell-specific g-aminobutyricacid type A receptor. Mol Pharmacol 1995;47:283–289.

41. Luddens H, Korpi ER. GABA antagonists differentiate betweenrecombinant GABAA/benzodiazepine receptor subtypes. J Neu-rosci 1995;15:6957–6962.

42. Banerjee PK, Olsen RW, Tillakaratne NJ, et al. Absence seizuresdecrease steroid modulation of t-[35S]butylbicyclophosphoro-thionate binding in thalamic relay nuclei. J Pharmacol Exp Ther1998;287:766–772.

43. Kim Y, Oh S. Changes of GABAA receptor binding and sub-unit mRNA level in rat brain by infusion of NOS inhibitor.Brain Res 2002;952:246–256.

44. DeLorey TM, Handforth A, Anagnostaras SG, et al. Mice lack-ing the beta3 subunit of the GABAA receptor have the epilepsyphenotype and many of the behavioral characteristics of An-gelman syndrome. J Neurosci 1998;18:8505–8514.

45. Fritschy JM, Brunig I. Formation and plasticity of GABAergicsynapses: physiological mechanisms and pathophysiological im-plications. Pharmacol Ther 2003;98:299–323.

46. Kittler JT, McAinsh K, Moss SJ. Mechanisms of GABAA re-ceptor assembly and trafficking. Mol Neurobiol 2002;26:251–268.

47. Liu F, Wan Q, Pristupa Z, et al. Direct protein-protein cou-pling enables cross-talk between dopamine D5 and gamma-amino butyric acid A receptors. Nature 2000;403:274–280.

48. Luscher B, Keller CA. Regulation of GABAA receptor traffick-ing, channel activity, and functional plasticity of inhibitory syn-apses. Pharmacol Ther 2004;102:195–221.


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Status epilepticus in mice deficient for succinate semialdehyde dehydrogenase: GABAA receptor–mediated mechanisms