T helper type 2-polarized invariant natural killer T cells reduce disease severity in acute intra-abdominal sepsis.

Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNK T) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of iNK T cells in their peripheral blood (as a percentage of their circulating T cells) compared to non-septic patients. We therefore investigated the role of iNK T cells in a mouse model of intra-abdominal sepsis (IAS). Our data show that iNK T cells are pathogenic in IAS, and that T helper type 2 (Th2) polarization of iNK T cells using the synthetic glycolipid OCH significantly reduces mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13 and reduction of several proinflammatory cytokines within the spleen, notably interleukin (IL)-17. Finally, we show that treatment of sepsis with OCH in mice is accompanied by significantly reduced apoptosis of splenic T and B lymphocytes and macrophages, but not natural killer cells. We propose that modulation of iNK T cell responses towards a Th2 phenotype may be an effective therapeutic strategy in early sepsis.

Cervical spine injuries and collar complications in severely injured paediatric trauma patients.

STUDY DESIGN: A retrospective registry review. OBJECTIVES: To determine the incidence of cervical spine (CS) injuries and collar complications in severely injured paediatric trauma patients. SETTING: Regional Trauma Centre, Children's Hospital. METHODS: A retrospective review of 365 paediatric severe trauma patients (0-17 years), defined as an Injury Severity Score (ISS)≥12, admitted to the paediatric intensive care unit (PICU). RESULTS: Clinically significant CS injuries occurred in 5% (n=18/365) of trauma patients, in 9% (n=13/149) of traumatic brain injury (TBI) patients and in 11% (n=6/56) of in-hospital trauma deaths. CS injuries were suspected before imaging in 33% (n=6/18) of patients based on either motor/sensory impairment or shock. CS injuries were deemed unstable in 61% (n=11/18) of patients. Patients with CS injuries had higher ISS, and longer PICU and hospital stays (P<0.05). CS collar complications occurred in 10% of patients, mainly identified by day 6 and consisting of either erythema or ulcers. Patients with CS collar complications were older and more likely to have TBI, lower Glasgow Coma Scale (GCS) scores, longer PICU and hospital stays, and increased days to CS clearance (P<0.05). Three CS X-rays, together with flexion/extension views, were used most frequently for CS clearance. CONCLUSION: CS injuries were prevalent in severely injured paediatric trauma patients, particularly in those with TBI and in nonsurvivors. CS collar complications were associated with a lower GCS and longer CS clearance times. Attention to CS collar management protocols and earlier CS clearance with computed tomography/magnetic resonance imaging in obtunded patients might reduce CS collar complications.

Arachidonic acid inhibits sodium currents and synaptic transmission in cultured striatal neurons.

In the striatum, dopamine generates arachidonic acid (AA) and induces synaptic depression. Here, we report that Na+ channels are a target for AA in both cultured and acutely isolated striatal neurons. AA depressed veratrine-induced Na+ influx and neurotransmitter release. Whole-cell voltage clamp revealed that peak Na+ currents are depressed, and steady-state inactivation shifts -15 mV in the presence of AA. Furthermore, inactivation was accelerated, and recovery from inactivation was delayed. These actions of AA were not produced by AA metabolites or protein kinase C activation. In addition, AA reduced both the amplitude and frequency of action potentials and depressed spontaneous inhibitory postsynaptic currents without affecting miniature inhibitory postsynaptic currents. These data suggest that AA modulates presynaptic, Na(+)-dependent action potentials, thereby contributing to striatal synaptic depression.

bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells.

In cultures of embryonic and adult mouse striatum, we previously demonstrated that EGF induces the proliferation of putative stem cells, which give rise to spheres of undifferentiated cells that can generate neurons and astrocytes. We report here that the spheres of undifferentiated cells contain mRNA and protein for the FGF receptor (FGFR1). Indirect immunocytochemistry demonstrated that many of the cells within the EGF-generated spheres were immunoreactive for FGFR1. Exogenous application of bFGF to the EGF-generated cells induced the proliferation of two progenitor cell types. The first, a bipotent progenitor cell, gave rise to cells with the antigenic and morphological properties of neurons and astrocytes; the other gave rise to cells with neuronal characteristics only. bFGF-generated cells with neuronal morphology exhibited electrophysiological properties indicative of immature central neurons. These results support the hypothesis that sequential actions of growth factors play a role in regulating the generation of neurons and astrocytes in the developing CNS.

The treatment of inclusion body myositis: a retrospective review and a randomized, prospective trial of immunosuppressive therapy.

We have sought to examine the response to immunosuppressive therapeutic intervention in inclusion body myositis (IBM) in a retrospective review of prior responses to therapy and in an open, randomized crossover trial. We collected information on the response to prior therapy on 25 patients, and for prospective therapy on 11 of these patients. All met criteria for a definite idiopathic inflammatory myopathy and had biopsy-proven IBM. Clinical and laboratory results were assessed by interviews of patients and by chart review in the retrospective trial. Manual muscle strength was assessed by a single trained observer; the patients' activities of daily living were assessed by questionnaire; and serum tests of muscle-associated enzymes were measured in the prospective trial. In the retrospective review, prednisone appeared to have been of some, albeit modest, clinical benefit in 10 of 25 (40%) patients. Other therapies, primarily azathioprine and methotrexate, also appeared to have halted the progression of weakness in 8 of 35 trials (23%). In the prospective study, combination therapy of oral azathioprine and methotrexate and a biweekly infusion of high-dose intravenous methotrexate with leucovorin rescue were given for 3 to 6 months in an open, crossover design. Both the oral and the intravenous regimens were clinically effective in some patients. There was clinical improvement in 3 trials, stabilization in 11 trials, and worsening in 5 trials, out of a total of 19 completed (22 intended) trials. The presence of active inflammation at entry into the prospective therapeutic protocol, either directly observed on muscle biopsy or indirectly indicated by serum creatine kinase level, may have been associated with clinical improvement. A complete laboratory response with normalization of creatine kinase and other muscle-associated enzymes did not, however, significantly predict clinical responsiveness in the prospective trial. In this first report, to our knowledge, of a prospective trial of immunosuppressive therapy for this disease, stabilization and even slight improvement of strength and functional abilities appeared to be achieved in some patients. We believe that prednisone and other immunosuppressive therapies were of modest benefit in about half of patients with inclusion body myositis, especially those with some evidence of active inflammation. Stabilization of an otherwise inexorably deteriorating course appears, therefore, to be an attainable goal in some patients with IBM.

A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups.

The IIM are a heterogeneous group of systemic rheumatic diseases which share the common features of chronic muscle weakness and mononuclear cell infiltrates in muscle. A number of classification schemes have been proposed for them, but none takes into consideration the marked immunologic, clinical, and genetic heterogeneity of the various clinical groups. We compared the usefulness of myositis-specific autoantibodies (anti-aminoacyl-tRNA synthetases, anti-SRP, anti-Mi-2 and anti-MAS) to the standard clinical categories (polymyositis, dermatomyositis, overlap myositis, cancer-associated myositis, and inclusion body myositis) in predicting clinical signs and symptoms, HLA types, and prognosis in 212 adult IIM patients. Although patients with inclusion body myositis (n = 26) differed in having significantly more asymmetric and distal weakness, falling, and atrophy than other patients, there were few other significant differences among the other clinical groups. In contrast, autoantibody status defined distinct sets of patients and each patient had only 1 myositis-specific autoantibody. Patients with anti-amino-acyl-tRNA synthetase autoantibodies (n = 47), compared to those without these antibodies, had significantly more frequent arthritis, fever, interstitial lung disease, and "mechanic's hands"; HLA-DRw52; higher mean prednisone dose at survey, higher proportion of patients receiving cytotoxic drugs, and higher death rates. Those with anti-signal recognition particle antibodies (n = 7) had increased palpitations; myalgias; DR5, DRw52; severe, refractory disease; and higher death rates. Patients with anti-Mi-2 antibodies (n = 10) had increased "V-sign" and "shawl-sign" rashes, and cuticular overgrowth; DR7 and DRw53; and a good response to therapy. The 2 patients with anti-MAS antibodies were the only ones with alcoholic rhabdomyolysis preceding myositis; both had insulin-dependent diabetes mellitus, and both had HLA-B60, -C3, -DR4, and -DRw53. These findings suggest that myositis-specific autoantibody status is a more useful guide than clinical group in assessing patients with myositis, and that specific associations of immunogenetics, immune responses, and clinical manifestations occur in IIM. Thus the myositis-specific autoantibodies aid in interpreting the diverse symptoms and signs of myositis patients and in predicting their clinical course and prognosis. We propose, therefore, that an adjunct classification of the IIM, based on the myositis-specific autoantibody status, be incorporated into future studies of their epidemiology, etiology, and therapy.

Drug therapy of the idiopathic inflammatory myopathies: predictors of response to prednisone, azathioprine, and methotrexate and a comparison of their efficacy.

PURPOSE: To identify factors associated with responses to treatment with prednisone, methotrexate, or azathioprine in patients with idiopathic inflammatory myopathy, and to compare the efficacy of these drugs. PATIENTS AND METHODS: Data were collected on 113 adult patients meeting criteria for definite idiopathic inflammatory myopathy in this retrospective cohort study. Patients were categorized as responding completely, partially, or not at all to each therapeutic trial based upon clinical and laboratory criteria. RESULTS: Clinical group, presence of certain myositis-specific autoantibodies, and time from disease onset to diagnosis influenced rates of complete clinical response to these therapeutic agents. Patients with inclusion body myositis responded comparatively poorly to prednisone and the other drugs: 43% had no clinical response to prednisone and none responded completely to any medication. Patients with autoantibodies to aminoacyl-tRNA synthetases or to signal recognition particle proteins were likely to respond partially, but not completely, to prednisone. No patient with a long delay to diagnosis (greater than 18 months) responded completely, compared with 34% of those with a short delay (less than 3 months). A patient's response to the first course of prednisone predicted subsequent responses to prednisone and to azathioprine better than response to methotrexate. Men responded to methotrexate better than women. Among certain subgroups of patients, responses to methotrexate were better than to either azathioprine or retreatment with prednisone. CONCLUSION: Determining the clinical group, autoantibody status, and time from disease onset to diagnosis of patients with myositis provides useful information in predicting clinical responses to therapy, and these factors should be considered in designing future therapeutic trials. Methotrexate therapy may be superior to either azathioprine or further steroid treatment alone in certain patients who do not respond completely to an initial adequate course of prednisone.

Diagnosis of inflammatory myopathy by percutaneous needle biopsy with demonstration of the focal nature of myositis.

Inflammatory myopathies are a group of acquired disorders with histologic features of inflammation and nonspecific myopathic changes in the muscle fibers. Up to 25% of patients with clinical features of polymyositis reportedly have no inflammatory changes in their muscle biopsy specimens, but the absence of inflammatory infiltrates does not exclude an inflammatory myopathy. However, whether the lack of inflammation is caused by sampling variation or by a total lack of demonstrable inflammation in a particular patient has been unclear in the literature. The authors diagnosed polymyositis in six patients who underwent percutaneous muscle biopsy using a Bergstrom needle. Through one skin incision, the needle was inserted into different areas within the muscle compartment, obtaining three or four concurrent specimens from each patient. In all cases of needle biopsy, adequate tissue was obtained for histochemical and electron microscopic examination. All patients tolerated the procedure well and resumed normal daily activities the morning after biopsy. Although we saw inflammatory changes in at least one biopsy specimen from each patient, one or more of the remaining specimens contained no evidence of inflammation. This illustrates that inflammatory infiltrates can be focal in polymyositis. Because a specific diagnosis of inflammatory myopathy cannot be made in the absence of demonstrable inflammation, the diagnostic yield of multiple percutaneous needle biopsy specimen is potentially higher than that of the traditional single biopsy specimen obtained with the open surgical method.

Adaptive responses to muscle lengthening and shortening in humans.

We tested the hypothesis that exercise training with maximal eccentric (lengthening) muscle actions results in greater gains in muscle strength and size than training with concentric (shortening) actions. Changes in muscle strength, muscle fiber size, and surface electromyographic (EMG) activity of the quadriceps muscle were compared after 36 sessions of isokinetic concentric (n = 8) or eccentric (n = 7) exercise training over 12 wk with use of a one-leg model. Eccentric training increased eccentric strength 3.5 times more (pre/post 46%, P < 0.05) than concentric training increased concentric strength (pre/post 13%). Eccentric training increased concentric strength and concentric training increased eccentric strength by about the same magnitude (5 and 10%, respectively, P > 0.05). Eccentric training increased EMG activity seven times more during eccentric testing (pre/post 86%, P < 0.05) than concentric training increased EMG activity during concentric testing (pre/post 12%). Eccentric training increased the EMG activity measured during concentric tests and concentric training increased the EMG activity measured during eccentric tests by about the same magnitude (8 and 11%, respectively, P > 0.05). Type I muscle fiber percentages did not change significantly, but type IIa fibers increased and type IIb fibers decreased significantly (P < 0.05) in both training groups. Type I fiber areas did not change significantly (P > 0.05), but type II fiber area increased approximately 10 times more (P < 0.05) in the eccentric than in the concentric group. It is concluded that adaptations to training with maximal eccentric contractions are specific to eccentric muscle actions that are associated with greater neural adaptation and muscle hypertrophy than concentric exercise.

Training cessation does not alter GLUT-4 protein levels in human skeletal muscle.

The purpose of this study was to determine whether short-term training cessation resulted in reduced GLUT-4 protein levels. Endurance- (n = 12, ET) and strength-trained (n = 12) individuals (ST) were examined before and after 14 days of training withdrawal. GLUT-4 content was determined from muscle biopsy samples of the gastrocnemius in ET and the vastus lateralis in ST. Insulin sensitivity (oral glucose tolerance test) was significantly (P < 0.05) reduced in ET and ST with training cessation. GLUT-4 content was unaltered (P > 0.05) in both groups (92 and 100% of trained values for ET and ST, respectively). In ET, citrate synthase activity decreased significantly (P < 0.05) with training withdrawal (41.0 +/- 3.6 vs. 30.6 +/- 2.8 mumol.g-1.min-1); in ST no change was evident. The decrement in insulin sensitivity with the cessation of endurance- or resistance-oriented activity is therefore not associated with a reduction in GLUT-4 protein content. Muscle oxidative capacity and GLUT-4 content do not coincide with the removal of endurance training.

Potential role of polyunsaturates in seizure protection achieved with the ketogenic diet.

Epilepsy is a serious neurological disease that responds to two very different treatments involving lipids. Clinically, it responds to a state of ketosis induced by a very high-fat 'ketogenic' diet. Experimentally, in vitro and in vivo models demonstrate that injection or infusion of free (non-esterified) polyunsaturates such as arachidonate and docosahexaenoate also reduces seizure susceptibility. In our experience, rats on a very high-fat ketogenic diet not only have mild-to-moderate ketosis, but also have raised serum free fatty acids. Some polyunsaturates, particularly linoleate and alpha-linolenate, are relatively easily beta-oxidized and are therefore ketogenic. We conclude that raised levels of free plasma polyunsaturates could contribute to the beneficial effect of the ketogenic diet in refractory epilepsy not only by helping sustain ketosis, but also by their own direct (though poorly defined) antiseizure effects.

Molecular detection of persistent Borrelia burgdorferi in a man with dermatomyositis.

A 40-year-old white man with a several year history of various immunologic disorders, including anti-Jo-1 autoantibody positive dermatomyositis, developed clinical Lyme disease after being biten by a tick. The patient was treated with oral tetracycline and his initial symptoms resolved; however, he suffered an exacerbation of his muscle disease which was difficult to control despite cytotoxic therapy. Antibiotic therapy was reinstituted after Borrelia burgdorferi was detected in the patient's peripheral blood leukocytes by the polymerase chain reaction (PCR). All serologic, T-cell stimulation, and western blot analyses, however, were negative. The patient's disease responded to oral ampicillin, probenecid therapy and concurrent cytotoxic therapy. Subsequent leukocyte PCR testing has been negative for the causative agent of Lyme disease. This case may provide an example of the in vivo immuno-modulatory effects of spirochetes in human autoimmune disease. In addition, this case emphasizes the potential clinical utility of PCR technology in evaluating the persistent sero-negative Lyme disease which may occur in immunocompromised individuals.

The effects of detraining on power athletes.

We investigated the effects of 14 d of resistive exercise detraining on 12 power athletes. In comparing performances pre- to post-detraining, there were no significant (P > 0.05) changes in free weight bench press (-1.7%), parallel squat (-0.9%), isometric (-7%) and isokinetic concentric knee extension force (-2.3%), and vertical jumping (1.2%). In contrast, isokinetic eccentric knee extension force decreased in every subject (-12%, P < 0.05). Post-detraining, the changes in surface EMG activity of the vastus lateralis during isometric, and isokinetic eccentric and concentric knee extension were -8.4%, -10.1%, and -12.7%, respectively (all P > 0.05). No significant changes occurred in knee flexion forces or EMGs (P > 0.05). Percentages of muscle fiber types and the Type I fiber area remained unchanged, but Type II fiber area decreased significantly by -6.4% (P < 0.05). Levels of plasma growth hormone (58.3%), testosterone (19.2%), and the testosterone to cortisol ratio (67.6%) increased, whereas plasma cortisol (-21.5%) and creatine kinase enzyme levels (-82.3%) decreased (all P < 0.05). Short-term resistive exercise detraining may thus specifically affect eccentric strength or the size of the Type II muscle fibers, leaving other aspects of neuromuscular performance uninfluenced. Changes in the hormonal milieu during detraining may be conducive to an enhanced anabolic process, but such changes may not materialize at the tissue level in the absence of the overload training stimulus.

Traumatic injury elicits JNK-mediated human astrocyte retraction in vitro.

Brain injury causes dysfunction of the blood-brain barrier (BBB). The BBB is comprised of perivascular astrocytes whose end-feet ensheath brain microvascular endothelial cells. We investigated trauma-induced morphological changes of human astrocytes (HA) and human cerebral microvascular endothelial cells (hCMEC/D3) in vitro, including the potential role of mitogen-activated protein kinase (MAPK) signal-transduction pathways. HA or hCMEC/D3 were grown on flexible culture membranes and subjected to single traumatic injury normalized to 20%, 30% or 55% membrane deformation. Cells were assayed for morphological changes (i.e. retraction) and MAPK phosphorylation and/or expression (c-Jun NH2-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38). HA retraction was rapidly elicited with a single traumatic injury (55% membrane deformation; p<0.01). Morphological recovery of HA was observed within 2h (p<0.05). Traumatic injuries increased phospho-JNK1/2 (p<0.05) in HA, indicating MAPK activation. Pre-treatment of HA with structurally distinct JNK inhibitors (25µM), either SP600125 or SU3327, reduced JNK phosphorylation (p<0.05) and trauma-induced HA retraction (P<0.05). In contrast to HA, traumatic injury failed to induce either morphological changes or MAPK activation in hCMEC/D3. In summary, traumatic injury induces JNK-mediated HA retraction in vitro, while sparing morphological changes in cerebral microvascular endothelial cells. Astrocyte retraction from microvascular endothelial cells in vivo may occur after brain trauma, resulting in cellular uncoupling and BBB dysfunction. JNK may represent a potential therapeutic target for traumatic brain injuries.

Insulin infusion via an intraosseous needle in diabetic ketoacidosis.

We report the successful management of a five-year-old child with severe diabetic ketoacidosis with dehydration, who received his initial resuscitative fluids and a continuous infusion of insulin via an intraosseous needle. The patient had presented to a remote community hospital and intravenous access could not be gained. The correction of hyperglycaemia and metabolic acidaemia was achieved at a rate comparable to intravenous therapy. No complications were observed. Although intraosseous access is well described in paediatric resuscitation guidelines, it is not mentioned in International Diabetes Society guidelines for the management of diabetic ketoacidosis. Alternatives to intravenous administration of insulin delivery recommended in such guidelines, such as the subcutaneous or intramuscular routes, may be less appropriate than the intraosseous route. This route can also allow resuscitation fluids and other drugs to be reliably administered in children with diabetic ketoacidosis and severe dehydration where intravenous access can not be attained. We suggest that the potential role of intraosseous access, when intravenous access can not be obtained, should be considered when management guidelines for paediatric diabetic ketoacidosis with dehydration are reviewed.

Lemierre's syndrome with septic shock caused by Fusobacterium necrophorum.

Fusobacterium necrophorum infections are rare. We report a 15-year-old male who presented with tachycardia, nausea, vomiting, diarrhoea and ankle pain. He rapidly deteriorated requiring ventilation and vasopressors. Imaging of his thorax showed airspace consolidation, pulmonary cavitations and empyema. The ankle required drainage of purulent material. A thrombus in his internal jugular vein (Lemierre's syndrome) and an abscess in his obturator internus were subsequently found. Fusobacterium necrophorum was identified in blood culture on day nine. The patient recovered with antibiotics and surgical interventions for empyema and septic arthritis. Fusobacterium necrophorum should be a suspected pathogen in septic shock complicated by metastatic abscess formation.

Low-threshold transient calcium current in rat hippocampal lacunosum-moleculare interneurons: kinetics and modulation by neurotransmitters.

Interneurons from the CA1 lacunosum-moleculare (L-M) region were isolated by trypsin-hyaluronidase treatment and mechanical trituration of the L-M. Interneurons isolated in this manner were multipolar with several dendritic processes and could be distinguished from CA1 pyramidal neurons. The properties of a low-threshold transient (LTT) Ca2+ current were investigated using whole-cell voltage-clamp techniques. The activation threshold of the LTT Ca2+ current was -60 mV, and the peak current, 100 +/- 9 pA (mean +/- SEM; n = 15), was observed at -30 mV. Ca2+ was the predominant charge carrier because the current was not affected by tetrodotoxin and was abolished in Ca(2+)-free external solution. Steady state inactivation was observed when the holding potential was positive to -100 mV, and the current was half-inactivated at -84 mV. Complete inactivation occurred at a holding potential of -60 mV. The time-to-peak of the current was highly voltage dependent and ranged from 10 msec at -60 mV to 4 msec at 0 mV. The time constant of inactivation was also voltage dependent and ranged from 27 msec at -60 mV to 12 msec at greater than -30 mV. Recovery from inactivation to 90% of maximum current occurred within 200 msec. L-M interneurons receive synaptic inputs from the septum that release ACh or GABA and from the raphe nuclei that release 5-HT. Carbachol, a nonhydrolyzable cholinergic agonist, and 5-HT quickly and reversibly increased the amplitude of the LTT Ca2+ current. Carbachol's actions were blocked by atropine, indicating that this effect was mediated by muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic-dependent plateau potential in hippocampal CA1 pyramidal neurons.

Cholinergic stimulation of the hippocampal formation results in excitation and/or seizure. We report here, using whole-cell patch-clamp techniques in the hippocampal slice (34-35 degrees C), a cholinergic-dependent slow afterdepolarization (sADP) and long-lasting plateau potential (PP). In the presence of 20 microM carbachol, action potential firing evoked by weak intracellular current injection elicited an sADP that lasted several seconds. Increased spike firing evoked by stronger depolarizing stimuli resulted in long-duration PPs maintained close to -20 mV. Removal of either Na+ or Ca2+ from the external media, intracellular Ca2+ ([Ca2+]i) chelation with 10 mM bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid, or the addition of 100 microM Cd2+ to the perfusate abolished both the sADP and PP. The sADP was depressed and the PP was abolished by either 10 microM nimodipine or 1 microM omega-conotoxin, whereas 1.2 microM tetrodotoxin was ineffective. The involvement of a Na+/Ca2+ exchanger was minimal because both the sADP and PP persisted after equimolar substitution of 50 mM Li+ for Na+ in the external media or reduction of the bath temperature to 25 degrees C. Finally in the absence of carbachol the sADP and PP could not be evoked when K+ channels were suppressed, suggesting that depression of K+ conductances alone was not sufficient to unmask the conductance. Based on these data, we propose that a Ca2+-activated nonselective cation conductance was directly enhanced by muscarinic stimulation. The sADP, therefore, represents activation of this conductance by residual [Ca2+]i, whereas the PP represents a novel regenerative event involving the interplay between high-voltage-activated Ca2+ channels and the Ca2+-activated nonselective cation conductance. This latter mechanism may contribute significantly to ictal depolarizations observed during cholinergic-induced seizures.

Serine/threonine protein phosphatases and synaptic inhibition regulate the expression of cholinergic-dependent plateau potentials.

We previously identified cholinergic-dependent plateau potentials (PPs) in CA1 pyramidal neurons that were intrinsically generated by interplay between voltage-gated calcium entry and a Ca(2+)-activated nonselective cation conductance. In the present study, we examined both the second-messenger pathway and the role of synaptic inhibition in the expression of PPs. The stimulation of m1/m3 cholinergic receptor subtypes and G-proteins were critical for activating PPs because selective receptor antagonists (pirenzepine, hexahydro-sila-difenidol hydrochloride, 4-diphenylacetoxy-N-methylpiperidine methiodide) and intracellular guanosine-5'-O-(2-thiodiphosphate) prevented PP generation in carbachol. Intense synaptic stimulation occasionally activated PPs in the presence of oxytremorine M, a cholinergic agonist with preference for m1/m3 receptors. PPs were consistently activated by synaptic stimulation only when oxytremorine M was combined with antagonists at both GABA(A) and GABA(B) receptors. These latter data indicate an important role for synaptic inhibition in preventing PP generation. Both intrinsically generated and synaptically activated PPs could not be elicited following inhibition of serine/threonine protein phosphatases by calyculin A, okadaic acid, or microcystin-L, suggesting that muscarinic-induced dephosphorylation is necessary for PP generation. PP genesis was also inhibited following irreversible thiophosphorylation by intracellular perfusion with ATP-gamma-S. These data indicate that the expression of cholinergic-dependent PPs requires protein phosphatase-induced dephosphorylation via G-protein-linked m1/m3 receptor(s). Moreover, synaptic inhibition via both GABA(A) and GABA(B) receptors normally prevents the synaptic activation of PPs. Understanding the regulation of PPs should provide clues to the role of this regenerative potential in both normal activity and pathophysiological processes such as epilepsy.

Elevated corticotropin releasing hormone/corticotropin releasing hormone-R1 expression in postmortem brain obtained from children with generalized epilepsy.

The corticotropin releasing hormone (CRH) system has been suggested to initiate seizure activity in the developing brain. However, human data to support this theory is lacking. In this study, we have demonstrated that the expression of CRH, CRH-binding protein, and CRH-R1 (a CRH membrane receptor) were significantly elevated in cortical tissue obtained from 6 children with generalized epilepsy (mean age 8.2+/-1.5 years) relative to age-matched controls (mean age 7.8+/-1.4 years). In contrast, no significant difference in the expression of CRH-R2 was observed. The advent of CRH-R1 receptor antagonists may prove useful as novel anticonvulsants.