Up-regulation of GABA(B) receptor mRNA and protein in the hippocampus of cocaine- and lidocaine-kindled rats.

To evaluate the effect of GABA(B) receptor in drug-kindled seizures, the gene expression of GABA(B) receptor in cocaine- and lidocaine-kindled rats was examined in this study. Rats were injected (i.p.) daily with cocaine (55 mg/kg) or lidocaine (65 mg/kg) until they experienced a motor seizure (kindling). After kindling, rats received a 1-day, 10-day, or 30-day drug washout period. The rats in the 1-day washout group were killed after the washout. Those in the 10-day and 30-day groups were challenged either with drug or saline, and killed 24 h later. Control rats were injected and challenged with saline. GABA(B)R1a, 1b and R2 mRNAs in discrete regions of brain were detected by in situ hybridization; GABA(B)R1a protein level was measured by Western blotting. Ninety percent of the cocaine-treated rats and 100% of the lidocaine-treated rats were kindled by day 12. Those rats responded to the challenge cocaine or lidocaine with a motor seizure after the 10-day and 30-day washout. GABA(B) receptor mRNA and protein levels in the hippocampus were significantly increased after the 1-day and 10-day washout, but not the 30-day washout. In addition, the levels in drug-treated and drug-challenged rats were significantly greater than those in drug-treated and saline-challenged rats after the 10-day washout. Those data suggest that changes of GABA(B) receptor gene expression could be a factor underlying the development of drug-kindled seizure, but not a necessary component for the maintenance of this phenomenon.

GABA-transaminase antisense oligodeoxynucleotide modulates cocaine- and pentylenetetrazol-induced seizures in mice.

The mechanism of action of many anticonvulsive agents is to increase the function of the GABAergic system. Inhibition of GABA-Transaminase (GABA-T), the degradative enzyme for GABA, increases GABA levels in the brain. In this study, antisense oligodeoxynucleotides (ASO) targeted at the start codon region of GABA-Transaminase mRNA were used to modify seizure activity. Mice were treated, by intracerebroventricular injection, with antisense oligos or appropriate controls. At various times after treatment, the animals were challenged with cocaine (70 mg/kg, i.p.) and observed for seizure activity. At 15 hours after treatment, 1.152 and 1.44 nmol antisense oligo blocked cocaine-induced seizures. There was no effect of antisense oligo 8 or 36 hours after treatment. In addition, treatment with 7.2 nmol antisense oligo prevented pentylenetetrazol-induced seizures. These data demonstrate the modulation of seizure threshold using antisense oligodeoxynucleotides to GABA-T.

Alterations in t-butylbicyclophosphorothionate binding in the brains of lidocaine-kindled rats.

This autoradiographic study examines regional GABAA receptors in lidocaine-kindled rats. [35S]t-Butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel, was used to radiolabel GABAA complexes. Male Sprague-Dawley rats were injected daily with lidocaine (65mg/kg, i.p.). Seizure activity was evaluated using the Racine Scale (Racine, 1972). The animals displayed a gradual increase in the indices and by day 20 greater than 50% were in stage 4 or 5. Regression of behavior was seen in half of the experimental group and this subgroup was considered 'compensated'. Autoradiographs were analyzed using a computer-based image analysis system. Several regions within the kindled group display a decrease in TBPS binding, including the subiculum, posterior lateral thalamic nuclei, the lateral hippocampus CA1, and the lateral hippocampus CA3. Conversely, within the compensated group these regions display normal or heightened TBPS binding. The data support the theory that alterations in the GABAA receptors are involved in the kindling model of epilepsy.

Elevated gamma-aminobutyric acid levels attenuate the metabolic response to bilateral ischemia.

Bilateral ischemia has been shown to alter the net brain levels of energy metabolites such as ATP, phosphocreatine, glucose, and glycogen. The amino acid neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on neural activity. The present studies were designed to evaluate the influence of elevated GABA levels on the metabolic sequelae of ischemia. The GABA transaminase inhibitor gamma-vinyl-GABA (GVG; vigabatrin) was administered to Mongolian gerbils before the production of a bilateral ischemic incident. GABA levels were elevated in all regions assayed. Levels of energy metabolites were also increased, an indication of reduced energy utilization. In control animals, in the absence of GVG, 1 min of bilateral ischemia produced decreases in the levels of all metabolites. In animals pretreated with GVG, the effects of 1 min of bilateral ischemia were attenuated. These data suggest that the level of ongoing activity may affect the response to an ischemic insult. Furthermore, GVG may have a clinical indication in reducing the effect of minor ischemic incidents.

Differential expression of gamma-aminobutyric acidA receptor subunits.

A 1.8-kilobase (kb) cDNA clone for a beta 1 subunit of the human gamma-aminobutyric acidA (GABAA) receptor has been isolated and sequenced. The longest open reading frame of the clone, pCLL610, contains nucleotide sequence encoding a portion of the putative signal sequence followed by 449 amino acids of the entire mature protein. The deduced amino acid sequence of pCLL610 differs from a recently described human beta 1 subunit by a single amino acid. The amino acid sequences of the human GABAA receptor beta 1 subunits share 98% identity with the beta 1 subunits of the bovine and rat GABAA receptor, with the majority of the differences occurring in the intracellular loop between the M3 and M4 transmembrane regions of the protein. A single 11-kb transcript is observed in Northern blots of poly(A)+ RNA isolated from rat brain probed with nick-translated pCLL610. In human brain, the pCLL610 probe recognized the 11-kb message, in addition to two other bands between 8 and 11-kb. Developmental studies of rat brain mRNA show that the message of the GABAA beta 1 subunit is highest at birth, rapidly decreases, and reaches adult levels of 5 to 7 days of age. This is in contrast to the development of the alpha 1 subunit, which is low from days 1 to 5 and increases to adult levels by days 14 to 25. Relative levels of the mRNA for the alpha 1 and beta 1 subunits vary among rat brain regions. The levels of mRNA for the alpha 1 subunit are similar in the cortex, hippocampus, and midbrain, whereas cerebellar levels are twice those in the cortex. The rank order of the relative amount of beta 1 subunit message is hippocampus greater than cortex = midbrain greater than cerebellum. These data, taken with our previous study of the alpha 1 subunits of the GABAA receptor, suggest that the differences in the distribution and regulation of the alpha 1 and beta 1 subunits may reflect a variety of subunit combinations forming the GABAA receptor. Heterogeneity in the GABAA receptor composition may provide a molecular basis for the diverse pharmacological properties associated with this receptor.

Effect of halide ions on t-[35S]butylbicyclophosphorothionate binding.

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.

Differential effects of iodide and chloride on allosteric interactions of the GABAA receptor.

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.

Transverse posterior element fractures associated with torsion.

Six examples of a previously undescribed class of transverse vertebral element fractures are presented. These fractures differ from Chance and Smith fractures and their variants in the following respects: (1) the etiology is torsion and not flexion; (2) there is neither distraction of posterior ring fragments nor posterior ligament tears; (3) in contrast to Chance and Smith fractures, extension of the fracture into the vertebral body is absent or minimal; (4) the transverse process of the lumbar vertebra is avulsed at its base with a vertical fracture, not split horizontally. These fractures occur in cervical, lumbar, and sacral vertebrae in normal or compromised areas of the spine.

Effect of chronic desmethylimipramine or electroconvulsive shock on selected brain and platelet neurotransmitter recognition sites.

Rats were treated with electroconvulsive shock (ECS), desmethylimipramine (DMI), ECS plus DMI, or diazepam. In vitro analyses showed that chronic ECS produced an elevated density of recognition sites for [3H]imipramine (IMI) in platelet membranes, but had no effect on membrane preparations derived from cortical tissue. A similar elevation in receptor binding was seen exclusively in platelets after chronic ECS plus DMI, whereas no effect was observed with DMI alone. Equilibrium dissociation constant (KD) values for [3H]IMI were also increased in platelet membranes from rats given chronic ECS or ECS plus DMI treatment. Chronic ECS or DMI administration produced a decreased density of beta-adrenergic recognition sites in frontal cortex and cerebellum as assessed by [3H]dihydroalprenolol (DHA) binding. The combination of ECS plus DMI produced a similar decrease. In addition, chronic diazepam administration produced a down-regulation of the beta-adrenergic receptor only in the cerebellum. These data provide evidence for the differential regulation of brain and peripheral neurotransmitter recognition sites.

Jogger's fracture and other stress fractures of the lumbo-sacral spine.

The posterior rings of the lower lumbo-sacral vertebrae are subject to stress fractures at any part - pedicle, pars, or lamina. The site of fracture is apparently determined by the axis of weight bearing. The three illustrative clinical examples cited include a jogger with a laminar fracture, a ballet dancer with pedicle fractures, and a nine-year-old boy with fractures of pars and lamina. Chronic low back pain is the typical complaint with stress fractures of the lower lumbo-sacral spine. Special imaging techniques are usually needed to demonstrate these lesions, including vertebral arch views, multi-directional tomography, and computed tomography (CT).

Maintenance of regional chemical integrity for energy metabolites in microwave heat inactivated mouse brain.

A method is described in which brain tissue from microwave heat inactivated mouse brain is prepared for microregional energy metabolite analysis. This method permits the sectioning of the tissue into sections 40 microns thick in which layers can be visualized and dissected. Results show no movement or leaching of metabolites from one microregion to another. This method therefore, permits the combined use of the two powerful neurochemical techniques of microwave irradiation for sacrifice and microregional analysis for energy metabolite determination.

5-Aryl-3-azabicyclo[3.2.0]heptan-6-one ketals, compounds with morphine-like analgesic activity.

A series of 5-aryl-3-azabicyclo[3.2.0] heptan -6-one ketals 6 were synthesized by hydride reduction of 1-aryl-4, 4-dimethoxy-1,2- cyclobutanedicarboximides 5. Imides 5 were obtained as the sole, regioselective products of the [2 + 2] photocycloaddition of 1,1- dimethoxyethylene to 2- arylmaleimides . The m-methoxyphenyl-N-methyl analogue 6a was demethylated to phenol 7 with EtSNa -DMF. Both 6a and 7 were similar to morphine in analgesic potency in rats and mice and showed physiological effects that were identical with those of morphine and that were completely reversed by naloxone. Compound 7 was identical with morphine in its ability to displace [3H]naloxone from homogenates of rat brain minus cerebellum. A molecular mechanics analysis of the m-methoxyphenyl analogue 6a showed that the nitrogen atom, the methoxyphenyl group, and the methoxyl oxygen cis to the phenyl group can be superimposed on the corresponding features of the morphine molecule, and perhaps this accounts for the observed opiate-receptor binding properties of 7.

The radiology of low back pain associated with posterior element lesions of the lumbar spine.

Chronic low back pain is frequent, ubiquitous, intermittent and usually difficult to categorize. Aside from the minority of cases with specific inflammatory or neoplastic causes, its etiology is usually vague and its radiographic evaluation obscure on routine films of the lumbar spine. For almost 50 years it has been known that disc disease was a significant etiologic factor. More recently, abnormal zygapophyseal joints have been pinpointed as specific areas of causation of low back pain. Other posterior element lesions associated with low back pain include facet fractures, interarticular isthmus defects, and laminar and pedicular fracture deformities. The radiographic demonstration of these lesions may require axially angled oblique views, vertebral arch views, and motion studies added to the standard projections. CT and apohyseal joint arthrography have been especially helpful.

The effect of rapid eye movement sleep deprivation on cortical beta-adrenergic receptors.

The relationship between rapid eye movement sleep deprivation (REMD) and rat beta-adrenergic receptors was evaluated. REMD was achieved using the platform method and verified by EEG and EMG recordings. Although the amount of REM sleep was diminished 90%, there was no alteration in either the number of binding sites or their affinity for [3H]-dihydroalprenolol. Periods of stress as well as recovery periods after REMD were also without effect on the cortical beta-adrenergic receptors. Thus no support is garnered for the interaction of REMD and the cortical beta-adrenergic receptor binding parameters, although REMD is sometimes used as a mode of therapy for depression and other antidepressives do in fact affect the beta-adrenergic system. The mechanism of REMD as a potential antidepressive therapy is yet to be elucidated.

The radiology of chronic neck pain: sequelae of occult traumatic lesions.

Occult traumatic lesions of the cervical spine usually involve the smaller, posterior elements of the vertebrae. Chronic neck pain associated with these lesions is intermittent and of variable severity, but with patterns that are fairly characteristic of the osseous lesions. Similarly, the long-term radiographic sequelae occur in fairly predictable patterns. The original osseous injury characteristically involves a fracture deformity and frequently some instability. There is a closed five point suspension system at each of the levels of the lower cervical spine, so that each of these five points is stressed whenever any one of them is deformed. Thus there follows bilateral hypertrophic changes and disc deterioration. Satellite changes also occur. The original deformity usually leads to rotation at the level of the lesion, cervical malalignment, and muscle imbalance. Therefrom the osseous and disc changes associated with chronic torticollis may occur, as well as distant instabilities.

Metabolic profile of hippocampal regions after bilateral ischemia and recovery.

Microanalysis methods were used to determine the effect of bilateral carotid occlusion on net levels of energy metabolites in discrete cellular regions of the hippocampus and dentate gyrus of the Mongolian gerbil. Glucose, glycogen, ATP and phosphocreatine levels were not decreased after one minute of bilateral occlusion. Three minutes of ischemia, however, produced a dramatic fall in net levels with no further decrease observed at fifteen minutes. Re-establishment of blood flow for five minutes after a fifteen minute ischemic episode resulted in replenishment of metabolites to pre-ischemic levels. Glucose was increased two to three times in sham-operated animals as compared to control (non-operated) animals. The increase was the result of the Na-pentobarbital anesthetic employed. The present data indicate that regions of the hippocampus and dentate gyrus respond in a uniform manner to bilateral occlusion of the carotid arteries. Further, most cells maintained enough viability to resume production of high-energy phosphate and carbohydrate metabolites.

Human brain receptor alterations in suicide victims.

A comparison was made of human postmortem muscarinic-cholinergic, beta-adrenergic and serotonergic (presynaptic) recognition sites in cortical tissues derived from suicide and homicide (control) victims. An elevation of 47% and 35% in the suicide group compared to controls was observed in receptor ligand binding for 3H-quinuclidinyl benzilate (QNB, muscarinic antagonist) and 3H-imipramine (IMI, a presynaptic serotonin marker), respectively. In contrast, no appreciable differences in 3H-dihydroalprenolol (DHA, beta-adrenergic antagonist) binding were observed between the two groups. Additionally, tissues from both groups of subjects were analyzed for tricyclic antidepressive agent (TAD) content. High performance liquid chromatographic (HPLC) tissue analysis revealed no detectable levels of tricyclic agents with an assay sensitivity of 50 picograms/mg tissue. The results presented herein demonstrate neurotransmitter-receptor alterations in suicide subjects compared to homicide (control) victims. The attendant roles of serotonergic and muscarinic-cholinergic processes in the psychobiology of suicide and depression are addressed.

The exaggerated supine oblique view of the cervical spine.

The technique of the 60 degree supine oblique view is described together with anatomic skeletal studies of this projection. The view is convenient for emergency room radiography and useful in other clinical radiography. The view separates widely the anterior and posterior portions of the vertebrae in a side to side projection. This makes for an elongated but detailed view of the articular processes, pedicles, and intervertebral foramina. In the cadaver specimen and clinically the view is shown to be useful in delineating fracture deformities of the articular process and visualizing constriction of the intervertebral foramen superiorly. Encroachment of the foramen superiorly is likely to compromise the emerging nerve root in this area.

Isoniazid induced seizures and cerebral cortical and cerebellar energy metabolism.

Isoniazid is a useful chemical convulsant in that metabolic events associated with the preseizure state can be easily examined. In the present study, net levels of glucose, glycogen, ATP, and phosphocreatine were measured using enzymatic techniques in control mice, and in those injected with isoniazid. Results from this study showed a differential effect of isoniazid on cells from the cerebral cortex and the cerebellum. In the preseizure stage, the high energy phosphates ATP and phosphocreatine were decreased in layer 1 and the pyramidal cell layer of the cerebral parietal cortex, but were unchanged in the cerebellum. At the onset of seizures, metabolites were decreased not only in cortical layers, but in the molecular layer and Purkinje cell rich layer of the cerebellum as well. The somewhat delayed response of the cerebellum emphasizes the differential nature of metabolism in various brain regions. Such a delay in cerebellar energy response to perturbation may be conducive to the seizure state. In another series of mice, either sodium valproate or clonazepam was administered prior to isoniazid, and metabolite studies repeated. Results showed that at a time when each anticonvulsant acted to eliminate overt seizure activity, the reduction in ATP and phosphocreatine was not as great as it was in seizing mice treated with isoniazid alone.