Severe and progressive neurotransmitter release aberrations in familial hemiplegic migraine type 1 Cacna1a S218L knock-in mice.

Familial hemiplegic migraine type 1 (FHM1) is caused by mutations in the CACNA1A gene, encoding neuronal presynaptic Ca(V)2.1 (P/Q-type) Ca(2+) channels. These channels mediate neurotransmitter release at many central synapses and at the neuromuscular junction (NMJ). Mutation S218L causes a severe neurological phenotype of FHM and, additionally, ataxia and susceptibility to seizures, delayed brain edema, and fatal coma after minor head trauma. Recently, we generated a Cacna1a S218L knock-in mutant mouse, displaying these features and reduced survival. A first electrophysiological study showed high susceptibility for cortical spreading depression, enhanced neuronal soma Ca(2+) influx, and at diaphragm NMJs, a considerable increase of neurotransmitter release. We here assessed the function of S218L knock-in NMJs at several muscle types in great detail. Pharmacological analyses using specific Ca(V) subtype-blocking toxins excluded compensatory contribution of non-Ca(V)2.1 channels. Endplate potentials were considerably broadened at many NMJs. High rate (40 Hz)-evoked acetylcholine release was slightly reduced; however, it was not associated with block of neurotransmission causing weakness, as assessed with grip strength measurements and in vitro muscle contraction experiments. The synaptopathy clearly progressed with age, including development of an increased acetylcholine release at low-rate nerve stimulation at physiological extracellular Ca(2+) concentration and further endplate potential broadening. Our results suggest enhanced Ca(2+) influx into motor nerve terminals through S218L-mutated presynaptic Ca(V)2.1 channels, likely because of the earlier reported negative shift of activation potential and reduced inactivation. Similar severe aberrations at central synapses of S218L mutant mice and humans may underlie or contribute to the drastic neurological phenotype.

High cortical spreading depression susceptibility and migraine-associated symptoms in Ca(v)2.1 S218L mice.

OBJECTIVE: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. METHODS: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. RESULTS: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. INTERPRETATION: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.

Automated segmentation of in vivo and ex vivo mouse brain magnetic resonance images.

Segmentation of magnetic resonance imaging (MRI) data is required for many applications, such as the comparison of different structures or time points, and for annotation purposes. Currently, the gold standard for automated image segmentation is nonlinear atlas-based segmentation. However, these methods are either not sufficient or highly time consuming for mouse brains, owing to the low signal to noise ratio and low contrast between structures compared with other applications. We present a novel generic approach to reduce processing time for segmentation of various structures of mouse brains, in vivo and ex vivo. The segmentation consists of a rough affine registration to a template followed by a clustering approach to refine the rough segmentation near the edges. Compared with manual segmentations, the presented segmentation method has an average kappa index of 0.7 for 7 of 12 structures in in vivo MRI and 11 of 12 structures in ex vivo MRI. Furthermore, we found that these results were equal to the performance of a nonlinear segmentation method, but with the advantage of being 8 times faster. The presented automatic segmentation method is quick and intuitive and can be used for image registration, volume quantification of structures, and annotation.

Enhanced circadian phase resetting in R192Q Cav2.1 calcium channel migraine mice.

OBJECTIVE: Mammalian circadian rhythms are driven by the circadian pacemaker of the suprachiasmatic nucleus (SCN) and are synchronized to the external 24-hour light/dark cycle. After advance time zone transitions (eastbound jet lag), overt circadian rhythms require several days to adjust. The retarded adaptation may protect against acute imbalance of different brain systems. Abrupt circadian rhythm changes may trigger migraine attacks, possibly because migraineurs have an inadequate adaptation mechanism. The novel R192Q knock-in migraine mouse model carries mutated Ca(v)2.1 calcium channels, causing increased presynaptic calcium influx and neurotransmitter release. We investigated whether these mice have an abnormal adjustment to phase advance shifts. METHODS: We examined phase resetting to 6-hour advance shifts of the light/dark cycle with behavioral and electroencephalographic recordings in R192Q and wild-type mice. We recorded excitatory postsynaptic currents in the SCN, and electrical impulse frequency in vitro and in vivo. RESULTS: R192Q mice showed a more than twofold enhanced adjustment of behavioral wheel-running activity and electroencephalographic patterns, as well as enhanced shifts of electrical activity of SCN neurons in vivo. No differences were found for in vitro recordings of the electrical impulse frequency in SCN slices. INTERPRETATION: R192Q migraine mice lack the physiological retardation in circadian adaptation to phase advance shifts. The opposite findings in vivo and in vitro exclude involvement of the retinal input pathway or the phase-shifting capacity of the SCN. Thus, the physiological inhibitory process appears to be mediated by Ca(v)2.1 channel-dependent afferent signaling from extra-SCN brain areas to the SCN.

Reduced ACh release at neuromuscular synapses of heterozygous leaner Ca(v)2.1-mutant mice.

Episodic ataxia type 2 (EA2) is an autosomal dominantly inherited neurological disorder. Patients have CACNA1A gene mutations resulting in truncation or single amino acid changes in the pore-forming subunit of Ca(v)2.1 (P/Q-type) Ca(2+) channels. These neuronal channels mediate synaptic neurotransmitter release. EA2 symptoms are thought to result from disturbed neurotransmission at cerebellar and neuromuscular synapses, caused by loss-of-function of Ca(v)2.1 channels. Heterozygous leaner (Ln/wt) mice, carrying a Cacna1a truncation mutation, as well as heterozygous Ca(v)2.1 null-mutant (KO/wt) mice may model synaptic aspects of EA2. We studied Ca(v)2.1-mediated acetylcholine (ACh) release at their neuromuscular junctions (NMJs) ex vivo. KO/wt mice did not show any ACh release abnormalities, not even at older age. However, Ln/wt mice had approximately 25% reduced spontaneous uniquantal ACh release and approximately 10% reduced nerve-stimulation evoked release, compared with wild-type. EA2 is treated with acetazolamide (AZA), but the pharmacotherapeutic mechanism is unknown. We tested the possibility of a direct influence on (mutant) presynaptic Ca(v)2.1 channel function by studying the acute effect of 50 muM AZA on ACh release at ex vivo NMJs of wild-type, KO/wt, and Ln/wt mice. No changes were found in any of the release parameters. Our results indicate that Ln-mutated Ca(v)2.1 channels at Ln/wt NMJs are either normally inserted in the presynaptic membrane but have reduced function, or that they inhibit wild-type channels by hampering their expression, trafficking, membrane insertion and/or function. In this respect Ln/wt NMJs may model EA2 synapses. Furthermore, AZA does not exert an acute, direct influence on the function of presynaptic (mutant) Ca(v)2.1 channels.

T(1) relaxation in in vivo mouse brain at ultra-high field.

Accurate knowledge of relaxation times is imperative for adjustment of MRI parameters to obtain optimal signal-to-noise ratio (SNR) and contrast. As small animal MRI studies are extended to increasingly higher magnetic fields, these parameters must be assessed anew. The goal of this study was to obtain accurate spin-lattice (T(1)) relaxation times for the normal mouse brain at field strengths of 9.4 and 17.6 T. T(1) relaxation times were determined for cortex, corpus callosum, caudate putamen, hippocampus, periaqueductal gray, lateral ventricle, and cerebellum and varied from 1651 +/- 28 to 2449 +/- 150 ms at 9.4 T and 1824 +/- 101 to 2772 +/- 235 ms at 17.6 T. A field strength-dependent increase of T(1) relaxation times is shown. The SNR increase at 17.6 T is in good agreement with the expected SNR increase for a sample-dominated noise regime.

Genetic models of migraine.

Migraine is a common, disabling, complex brain disorder, presenting in attacks that may have up to 3 phases: a prodromal phase, the aura phase, and the headache phase. The pathogenesis of the aura and headache phases is reasonably well understood, but the mechanism by which migraine attacks are triggered is unknown. Most likely, migraineurs have a genetically determined reduced threshold for migraine triggers. Identifying "threshold genes" and deciphering their function will help to unravel the triggering mechanisms for migraine attacks. Familial hemiplegic migraine is a rare monogenic subtype of migraine with aura. Three genes have been identified for familial hemiplegic migraine. Recently, knock-in mice carrying human pathogenic FHM1 mutations were generated, which show behavioral, electrophysiological, and neurobiological characteristics in line with prevailing views of migraine physiological processes. Genetic migraine models will be useful in unraveling the triggering mechanisms for migraine attacks and in identifying novel migraine prophylactic targets and therapies.

Severely impaired neuromuscular synaptic transmission causes muscle weakness in the Cacna1a-mutant mouse rolling Nagoya.

The ataxic mouse rolling Nagoya (RN) carries a missense mutation in the Cacna1a gene, encoding the pore-forming subunit of neuronal Ca(v)2.1 (P/Q-type) Ca2+ channels. Besides being the predominant type of Ca(v) channel in the cerebellum, Ca(v)2.1 channels mediate acetylcholine (ACh) release at the peripheral neuromuscular junction (NMJ). Therefore, Ca(v)2.1 dysfunction induced by the RN mutation may disturb ACh release at the NMJ. The dysfunction may resemble the situation in Lambert-Eaton myasthenic syndrome (LEMS), in which autoantibodies target Ca(v)2.1 channels at NMJs, inducing severely reduced ACh release and resulting in muscle weakness. We tested neuromuscular function of RN mice and characterized transmitter release properties at their NMJs in diaphragm, soleus and flexor digitorum brevis muscles. Clinical muscle weakness and fatigue were demonstrated using repetitive nerve-stimulation electromyography, grip strength testing and an inverted grid hanging test. Muscle contraction experiments showed a compromised safety factor of neuromuscular transmission. In ex vivo electrophysiological experiments we found severely impaired ACh release. Compared to wild-type, RN NMJs had 50-75% lower nerve stimulation-evoked transmitter release, explaining the observed muscle weakness. Surprisingly, the reduction in evoked release was accompanied by an approximately 3-fold increase in spontaneous ACh release. This synaptic phenotype suggests a complex effect of the RN mutation on different functional Ca(v)2.1 channel parameters, presumably with a positive shift in activation potential as a prevailing feature. Taken together, our studies indicate that the gait abnormality of RN mice is due to a combination of ataxia and muscle weakness and that RN models aspects of the NMJ dysfunction in LEMS.

Redundancy of Cav2.1 channel accessory subunits in transmitter release at the mouse neuromuscular junction.

Ca(v)2.1 (P/Q-type) channels possess a voltage-sensitive pore-forming alpha(1) subunit that can associate with the accessory subunits alpha(2)delta, beta and gamma. The primary role of Ca(v)2.1 channels is to mediate transmitter release from nerve terminals both in the central and peripheral nervous system. Whole-cell voltage-clamp studies in in vitro expression systems have indicated that accessory channel subunits can have diverse modulatory effects on membrane expression and biophysical properties of Ca(v)2.1 channels. However, there is only limited knowledge on whether similar modulation also occurs in the specific presynaptic environment in vivo and, hence, whether accessory subunits influence neurotransmitter release. Ducky, lethargic and stargazer are mutant mice that lack functional alpha(2)delta-2, beta(4) and gamma(2) accessory Ca(v) channel subunits, respectively. The neuromuscular junction (NMJ) is a peripheral synapse, where transmitter release is governed exclusively by Ca(v)2.1 channels, and which can be characterized electrophysiologically with relative experimental ease. In order to investigate a possible synaptic influence of accessory subunits in detail, we electrophysiologically measured acetylcholine (ACh) release at NMJs of these three mutants. Surprisingly, we did not find any changes compared to wild-type littermates, other than a small reduction (25%) of evoked ACh release at ducky NMJs. This effect is most likely due to the approximately 40% reduced synapse size, associated with the reduced size of ducky mice, rather than resulting directly from reduced Ca(v)2.1 channel function due to alpha(2)delta-2 absence. We conclude that alpha(2)delta-2, beta(4), and gamma(2) accessory subunits are redundant for the transmitter release-mediating function of presynaptic Ca(v)2.1 channels at the mouse NMJ.

Conditional inactivation of the Cacna1a gene in transgenic mice.

Ca(v)2.1 (P/Q-type) voltage-gated calcium channels play an important role in neurotransmitter release at many brain synapses and at the neuromuscular junction. Mutations in the CACNA1A gene, encoding the pore forming alpha(1) subunit of Ca(v)2.1 channels, are associated with a wide spectrum of neurological disorders. Here we generated mice with a conditional, floxed, Cacna1a allele without any overt phenotype. Deletion of the floxed Cacna1a allele resulted in ataxia, dystonia, and lethality during the fourth week, a severe phenotype similar to conventional Ca(v)2.1 knockout mice. Although neurotransmitter release at the neuromuscular junction was not affected in the conditional mice, homozygous deletion of the floxed allele caused an ablation of Ca(v)2.1 channel-mediated neurotransmission that was accompanied by a compensatory upregulation of Ca(v)2.3 (R-type) channels at this synapse. Pharmacological inhibition of Ca(v)2.1 channels is possible, but the contributing cell-types and time windows relevant to the different Ca(v)2.1-related neurological disorders can only be reliably determined using Cacna1a conditional mice.

Compensatory contribution of Cav2.3 channels to acetylcholine release at the neuromuscular junction of tottering mice.

Tottering (Tg) mice carry the mutation P601L in their Cacna1a encoded Cav2.1 channels. Transmitter release at the wild-type neuromuscular junction (NMJ) is almost exclusively mediated by Cav2.1 channels, and we used this model synapse to study synaptic consequences of the Tg mutation. With electrophysiology, and using subtype-specific Cav2 channel-blocking toxins, we assessed a possible compensatory contribution of non-Cav2.1 channels to evoked acetylcholine (ACh) release at Tg NMJs. Release was reduced by approximately 75% by the Cav2.1 channel blocker omega-agatoxin-IVA, which was less than the approximately 95% reduction observed in wild-type. Release at Tg NMJs, but not at wild-type synapses, was reduced by approximately 15% by SNX-482, a Cav2.3 channel blocker. No Cav2.2 channel involvement was found. Probably, there is a small reduction in functional presynaptic Cav2.1 channels at Tg NMJs, which is compensated for by Cav2.3 channels. The remaining Cav2.1 channels are likely to convey enlarged Ca2+ flux, because evoked ACh release at Tg NMJs, at low extracellular Ca2+ concentration, was approximately sixfold higher than at wild-type NMJs. This is the first report of compensatory expression of non-Cav2.1 channels at NMJs of mice with a single amino acid change in Cav2.1.

A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression.

Migraine is a common, disabling, multifactorial, episodic neurovascular disorder of unknown etiology. Familial hemiplegic migraine type 1 (FHM-1) is a Mendelian subtype of migraine with aura that is caused by missense mutations in the CACNA1A gene that encodes the alpha(1) subunit of neuronal Ca(v)2.1 Ca(2+) channels. We generated a knockin mouse model carrying the human pure FHM-1 R192Q mutation and found multiple gain-of-function effects. These include increased Ca(v)2.1 current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and, in the intact animal, a reduced threshold and increased velocity of cortical spreading depression (CSD; the likely mechanism for the migraine aura). Our data show that the increased susceptibility for CSD and aura in migraine may be due to cortical hyperexcitability. The R192Q FHM-1 mouse is a promising animal model to study migraine mechanisms and treatments.

An external audit of laparoscopic cholecystectomy performed in medical treatment facilities of the department of Defense.

OBJECTIVE: This study provided an objective survey by an outside auditing group of a large, complete patient population undergoing laparoscopic cholecystectomies, determined the frequency of complications, especially bile duct injuries, and presented a system for classifying and comparing the severity of bile duct injuries. SUMMARY BACKGROUND DATA: This is the first study of laparoscopic cholecystectomy to encompass a large and complete patient population and to be based on objectively collected data rather than self-reported data. The Civilian External Peer Review Program (CEPRP) of the Department of Defense health care system conducted a retrospective study of 5642 patients who underwent laparoscopic cholecystectomies at 89 military medical treatment facilities from July 1990 through May 1992. METHODS: The study sample consisted of the complete records of 5607 (99.38%) of the 5642 laparoscopic cholecystectomy patients. RESULTS: Of the sample, 6.87% of patients experienced complications within 30 days of surgery, 0.57% sustained bile duct injuries, and 0.5% sustained bowel injuries. Among 5154 patients whose procedures were completed laparoscopically, 5.47% experienced complications. Laparoscopic procedures were converted to open cholecystectomies in 8.08% of cases. Intraoperative cholangiograms were attempted in 46.5% of cases and completed in 80.59% of those attempts. There were no intraoperative deaths; 0.04% of the patients died within 30 days of surgery. CONCLUSIONS: The frequency of complications found in this study is comparable to the frequency of complications reported in recent large civilian studies and earlier, smaller studies. The authors present a system for classifying bile duct injuries, which is designed to standardize references to such injuries and allow for accurate comparison of bile duct injuries in the future.

Intravagal paraganglioma: report of a case and a discussion of vascular parapharyngeal masses.

A case of intravagal paraganglioma that appeared as a parapharyngeal mass 1 year after tonsillectomy is discussed. The diagnosis of this lesion was based on angiographic findings, and subtraction views provided the best detail of the extent and nature of the mass. Excision was made nearly bloodless by initial ligation of the external carotid artery, followed by the application of direct pressure to the mass during dissection. The diagnosis of parapharyngeal vascular masses is discussed, and the pertinent literature is reviewed.

Reperfusion seizures after innominate endarterectomy.

Seizures occurring after carotid revascularization are an uncommon complication that has received increasing recognition. The development of postoperative seizures has been estimated to occur in 0.4% to 1.0% of all carotid endarterectomies. Patients with high-grade carotid arterial stenoses appear to be at highest risk for postoperative seizures, which are thought to be due to cortical hyperperfusion states. We report two cases of focal motor seizures occurring after innominate endarterectomy, demonstrating that this complication is not limited to carotid arterial surgery.

Surgical treatment of occlusive subclavian artery disease.

Thirty-eight patients underwent operations for subclavian artery stenosis over a 12-year period. Nineteen patients had neurologic symptoms and 12 had claudication or ischemia. The classic subclavian steal syndrome was rare. Twenty patients underwent carotid subclavian bypass and 14 underwent subclavian endarterectomy using a cervical or thoracic approach. Follow-up data were available in 35 of 38 patients (average follow-up 36 months). Thirteen of the 14 patients who underwent endarterectomy remained asymptomatic and showed no evidence of restenosis (average follow-up 53 months). Of the 20 patients who had carotid subclavian bypass, Dacron was the graft material used in eight patients and autogenous saphenous vein was used in 12. Thrombosis occurred in five of 12 saphenous vein grafts, two immediately after operation, one at 2 months and two at 10 months. An anastomotic stenosis was identified and corrected in one patient 38 months postoperatively. Recurrent or persistence of symptoms was directly related to graft failure. Thrombosis or stenosis did not occur in any of the Dacron grafts. All patients had some relief of symptoms. Subclavian endarterectomy or carotid subclavian bypass with Dacron gave excellent long-term results. Autogenous saphenous vein were unsatisfactory for these short bypasses.

Chronic common carotid occlusion.

Twelve patients with chronic common carotid artery (CCA) occlusion were studied. There were 8 patients with TIAs (3 hemispheric and 5 vertebral-basilar), one with a completed stroke, and 3 were asymptomatic. The ipsilateral internal carotid artery (ICA) was confirmed patent in 6 patients at the time of operation, although angiograms had demonstrated patency in only two. The ipsilateral external carotid artery (ECA) was patent in all but one patient. Arterial reconstructions were done on 7 patients. The carotid bifurcation was revascularized by subclavian-to-carotid bypass grafts in 5 patients, 3 with vertebral-basilar (V-B) TIAs, one with hemispheric TIAs, and one with a completed stroke. Vertebral revascularization was done on 2 patients, one with V-B TIAs and one who was asymptomatic. All revascularized patients had satisfactory results. Symptomatic patients can be treated by cerebral revascularization through either the ICA, if patent, the ECA via the ophthalmic collaterals, or through the vertebrals when hypoperfused. The ICA is preferentially revascularized and exploration often reveals a patent ICA that was not visualized angiographically. Recently, directional Doppler studies have proved useful in determining ICA patency.

Failure of Doppler ultrasound in the early assessment of blood flow in polytetrafluoroethylene grafts.

PTFE is widely used as an arterial and venous graft. The assessment of patency and flow through vascular grafts has been facilitated by the use of Doppler blood flow measurement instruments. However PTFE grafts do not predictably transmit the ultrasound beam used in these instruments in the operative and early postoperative period. Consequently Doppler flow measurements over PTFE grafts are unreliable and may confuse the early assessment of operative results.