An adaptable, reusable, and light implant for chronic Neuropixels probes.

Electrophysiology has proven invaluable to record neural activity, and the development of Neuropixels probes dramatically increased the number of recorded neurons. These probes are often implanted acutely, but acute recordings cannot be performed in freely moving animals and the recorded neurons cannot be tracked across days. To study key behaviors such as navigation, learning, and memory formation, the probes must be implanted chronically. An ideal chronic implant should (1) allow stable recordings of neurons for weeks; (2) be light enough for use in mice; (3) allow reuse of the probes after explantation. Here, we present the "Apollo Implant", an open-source and editable device that meets these criteria and accommodates up to two Neuropixels 1.0 or 2.0 probes. The implant comprises a "payload" module that is attached to the probe and is recoverable, and a "docking" module that is cemented to the skull. The design is adjustable, making it easy to change the distance between probes, the angle of insertion, and the depth of insertion. We tested the implant across seven labs in head-fixed mice, freely moving mice, and freely moving rats. The number of neurons recorded across days was stable, even after repeated implantations of the same probe. The Apollo implant provides an inexpensive, lightweight, and flexible solution for reusable chronic Neuropixels recordings.

Preparation of dissociated mouse primary neuronal cultures from long-term cryopreserved brain tissue.

BACKGROUND: Dissociated primary neuronal cultures are widely used as a model system to investigate the cellular and molecular properties of diverse neuronal populations and mechanisms of action potential generation and synaptic transmission. Typically, rodent primary neuronal cultures are obtained from freshly-dissociated embryonic or postnatal brain tissue, which often requires intense animal husbandry. This can strain resources when working with genetically modified mice. NEW METHOD: Here we describe an experimental protocol for frozen storage of mouse hippocampi, which allows fully functional dissociated primary neuronal cultures to be prepared from cryopreserved tissue. RESULTS: We show that thawed hippocampal neurons have functional properties similar to those of freshly dissociated neurons, including neuronal morphology, excitability, action potential waveform and synaptic neurotransmitter release, even after cryopreservation for several years. COMPARISON TO THE EXISTING METHODS: In contrast to the existing methods, the protocol described here allows for efficient long-term storage of samples, allowing researchers to perform functional experiments on neuronal cultures from brain tissue collected in other laboratories. CONCLUSIONS: We anticipate that this method will facilitate collaborations among laboratories based at distant locations and will thus optimise the use of genetically modified mouse models, in line with the 3Rs (Replacement, Reduction and Refinement) recommended for scientific use of animals in research.

Changes in the severity and subtype of Guillain-Barré syndrome admitted to a specialist Neuromedical ICU over a 25 year period.

We report a retrospective review of 110 patients with acute Guillain-Barré syndrome (GBS) admitted to a specialised intensive care unit (ICU) in a tertiary referral centre over a 25 year period, the start of which coincided with the widespread introduction of plasma exchange (PE) and intravenous immunoglobulin (IVIG). The results were analysed by comparing 52 patients admitted in the first decade (1991-2000; Group 1) with 58 patients admitted between 2001-2014 (Group 2). Patients in both groups were comparable with respect to age and sex, and had a similar incidence and range of ICU complications. They received a comparable range of immunomodulatory treatments including IVIG and PE. However, the delay from presentation to referral to the tertiary ICU was longer in patients in Group 2. They also required mechanical ventilation for a longer duration, and had longer ICU and hospital stays. In Group 2, there was a higher incidence of axonal neuropathy (51%, compared to 24% in Group 1). Despite the longer delay to referral, the prevalence of axonal neuropathy and the duration of ventilation, overall mortality showed a downward trend (Group 1: 13.5%; Group 2: 5.2%). There was no late mortality in either group after step-down to neuro-rehabilitation or following discharge home or to the referring hospital. The rehabilitation outcomes were similar. This data show a shift in the pattern of referral to a tertiary referral ICU between the first and second decades following the wider availability of IVIG and PE for the treatment of GBS. The possible causes and implications of these findings are discussed.

Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801.

It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1(ΔPV) mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1(ΔPV) mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1(ΔPV)mice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1(ΔPV)mice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1(ΔPV)mice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease.

Genetic neurological channelopathies: molecular genetics and clinical phenotypes.

Evidence accumulated over recent years has shown that genetic neurological channelopathies can cause many different neurological diseases. Presentations relating to the brain, spinal cord, peripheral nerve or muscle mean that channelopathies can impact on almost any area of neurological practice. Typically, neurological channelopathies are inherited in an autosomal dominant fashion and cause paroxysmal disturbances of neurological function, although the impairment of function can become fixed with time. These disorders are individually rare, but an accurate diagnosis is important as it has genetic counselling and often treatment implications. Furthermore, the study of less common ion channel mutation-related diseases has increased our understanding of pathomechanisms that is relevant to common neurological diseases such as migraine and epilepsy. Here, we review the molecular genetic and clinical features of inherited neurological channelopathies.

Myasthenia gravis--treatment of acute severe exacerbations in the intensive care unit results in a favourable long-term prognosis.

BACKGROUND AND PURPOSE: Acute severe exacerbations of myasthenia gravis (MG) are common in both early and late onset MG. We wished to examine the current management in the intensive care unit (ICU) of severe exacerbations of MG and to study the long-term prognosis of MG following discharge from the ICU. METHODS: We retrospectively reviewed the medical records of all patients admitted to a specialist neuro-ICU with acute exacerbations of MG over a 12-year period. RESULTS: We identified 38 patients. Over 60% were over the age of 50 years, and MG was newly diagnosed in over 40%. Intubation was required in 63%, and over 90% of patients were treated with prednisolone and/or intravenous immunoglobulin. Four patients died in hospital. The remainder of patients were followed up for a mean of 4 years, and the majority were either asymptomatic or had mild symptoms of MG at clinical review. CONCLUSIONS: Despite the significant morbidity and mortality associated with severe exacerbations of MG, specialized neurointensive care can result in a good long-term prognosis in both early- and late-onset MG.

Thymectomy: role in the treatment of myasthenia gravis.

Thymectomy is a frequently used treatment for myasthenia gravis (MG) and is virtually always indicated in MG patients who have a thymoma. However, the evidence for thymectomy in non-thymomatous MG remains less certain-no randomised controlled trials have been published to date, although one is currently underway. We reviewed the management and clinical outcome of patients with MG who underwent thymectomy over a 12 year period. Eighty-nine patients who underwent transsternal thymectomy were identified. A thymoma was identified on histology in 24 %, whereas 48, 9 and 19 % had hyperplastic, atrophic and normal thymic histology, respectively. One patient developed post operative myasthenic crisis but generally the procedure was well tolerated. Outcome was favourable for the majority of patients, with 34 % achieving complete stable remission (CSR) and an additional 33 % achieving pharmacological remission. Moreover, steroid requirements fell progressively during follow-up. Patients with a hyperplastic gland had a significantly greater chance of achieving CSR compared to other histological subtypes and the incidence of CSR increased with a longer duration of follow-up. Thymectomy for MG is generally safe and well tolerated and is associated with a sustained improvement of symptoms in the majority of patients.

Myasthenia gravis and neuromyelitis opica: A causal link.

Neuromyelitis Optica (NMO) and Myasthenia Gravis (MG) are rare antibody mediated disorders of the central nervous system (CNS) and neuromuscular junction (NMJ) respectively. Both diseases are predominantly mediated by IgG1 antibodies that activate complement. There have been increasing reports of patients who develop both disorders. Given the rarity of both diseases it would seem that these occurrences are not purely coincidental. There is heterogeneity between the cases described in the literature but common trends are observed in patients who develop both disorders. Most patients described are female. Typically the MG precedes the NMO and the majority of patients have undergone thymectomy. Generally, the symptoms of MG are mild but the NMO tends to follow a more aggressive clinical course. The pathogenesis of NMO in combination with MG is unknown, but thymectomy has been implicated in a subset of patients. We present the case of a female patient who developed NMO on a background of sero-positive MG and discuss the relevant literature.

Nongenetic factors influence severity of episodic ataxia type 1 in monozygotic twins.

OBJECTIVE: Episodic ataxia type 1 (EA1) is a monogenic channelopathy caused by mutations of the potassium channel gene KCNA1. Affected individuals carrying the same mutation can exhibit considerable variability in the severity of ataxia, neuromyotonia, and other associated features. We investigated the phenotypic heterogeneity of EA1 in 2 sets of identical twins to determine the contribution of environmental factors to disease severity. One of the mutations was also found in a distantly related family, providing evidence of the influence of genetic background on the EA1 phenotype. METHODS: We evaluated 3 families with an EA1 phenotype, 2 of which included monozygotic twins. We sequenced the KCNA1 gene and studied the biophysical consequences of the mutations in HEK cells. RESULTS: We identified a new KCNA1 mutation in each pair of twins. Both pairs reported striking differences in the clinical severity of symptoms. The F414S mutation identified in one set of twins also occurred in a distantly related family in which seizures complicated the EA1 phenotype. The other twins had an R307C mutation, the first EA1 mutation to affect an arginine residue in the voltage-sensor domain. Both mutants when expressed exerted a dominant-negative effect on wild-type channels. CONCLUSION: These results broaden the range of KCNA1 mutations and reveal an unexpectedly large contribution of nongenetic factors to phenotypic variability in EA1. The occurrence of epilepsy in 1 of 2 families with the F414S mutation suggests an interplay of KCNA1 with other genetic factors.

Large scale calcium channel gene rearrangements in episodic ataxia and hemiplegic migraine: implications for diagnostic testing.

BACKGROUND: Episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are autosomal dominant disorders characterised by paroxysmal ataxia and migraine, respectively. Point mutations in CACNA1A, which encodes the neuronal P/Q-type calcium channel, have been detected in many cases of EA2 and FHM1. The genetic basis of typical cases without CACNA1A point mutations is not fully known. Standard DNA sequencing methods may miss large scale genetic rearrangements such as deletions and duplications. The authors investigated whether large scale genetic rearrangements in CACNA1A can cause EA2 and FHM1. METHODS: The authors used multiplex ligation dependent probe amplification (MLPA) to screen for intragenic CACNA1A rearrangements. RESULTS: The authors identified five previously unreported large scale deletions in CACNA1A in seven families with episodic ataxia and in one case with hemiplegic migraine. One of the deletions (exon 6 of CACNA1A) segregated with episodic ataxia in a four generation family with eight affected individuals previously mapped to 19p13. In addition, the authors identified the first pathogenic duplication in CACNA1A in an index case with isolated episodic diplopia without ataxia and in a first degree relative with episodic ataxia. CONCLUSIONS: Large scale deletions and duplications can cause CACNA1A associated channelopathies. Direct DNA sequencing alone is not sufficient as a diagnostic screening test.

Voltage sensor charge loss accounts for most cases of hypokalemic periodic paralysis.

BACKGROUND: Several missense mutations of CACNA1S and SCN4A genes occur in hypokalemic periodic paralysis. These mutations affect arginine residues in the S4 voltage sensors of the channel. Approximately 20% of cases remain genetically undefined. METHODS: We undertook direct automated DNA sequencing of the S4 regions of CACNA1S and SCN4A in 83 cases of hypokalemic periodic paralysis. RESULTS: We identified reported CACNA1S mutations in 64 cases. In the remaining 19 cases, mutations in SCN4A or other CACNA1S S4 segments were found in 10, including three novel changes and the first mutations in channel domains I (SCN4A) and III (CACNA1S). CONCLUSIONS: All mutations affected arginine residues, consistent with the gating pore cation leak hypothesis of hypokalemic periodic paralysis. Arginine mutations in S4 segments underlie 90% of hypokalemic periodic paralysis cases.

Chloride channel myotonia: exon 8 hot-spot for dominant-negative interactions.

Myotonia congenita (MC) is the commonest genetic skeletal muscle ion channelopathy. It is caused by mutations in CLCN1 on chromosome 7q35, which alter the function of the major skeletal muscle voltage-gated chloride channel. Dominant and recessive forms of the disease exist. We have undertaken a clinical, genetic and molecular expression study based upon a large cohort of over 300 UK patients. In an initial cohort of 22 families, we sequenced the DNA of the entire coding region of CLCN1 and identified 11 novel and 11 known mutations allowing us to undertake a detailed genotype-phenotype correlation study. Generalized muscle hypertrophy, transient weakness and depressed tendon reflexes occurred more frequently in recessive than dominant MC. Mild cold exacerbation and significant muscle pain were equally common features in dominant and recessive cases. Dominant MC occurred in eight families. We noted that four newly identified dominant mutations clustered in exon 8, which codes for a highly conserved region of predicted interaction between the CLC-1 monomers. Expressed in Xenopus oocytes these mutations showed clear evidence of a dominant-negative effect. Based upon the analysis of mutations in this initial cohort as well as a review of published CLCN1 mutations, we devised an exon hierarchy analysis strategy for genetic screening. We applied this strategy to a second cohort of 303 UK cases with a suspected diagnosis of MC. In 23 individuals, we found two mutations and in 86 individuals we identified a single mutation. Interestingly, 40 of the cases with a single mutation had dominant exon 8 mutations. In total 48 individuals (from 34 families) in cohort 1 and 2 were found to harbour dominant mutations (37% of mutation positive individuals, 30% of mutation positive families). In total, we have identified 23 new disease causing mutations in MC, confirming the high degree of genetic heterogeneity associated with this disease. The DNA-based strategy we have devised achieved a genetic diagnosis in 36% of individuals referred to our centre. Based on these results, we propose that exon 8 of CLCN1 is a hot-spot for dominant mutations. Our molecular expression studies of the new exon 8 mutations indicate that this region of the chloride channel has an important role in dominant negative interactions between the two chloride channel monomers. Accurate genetic counselling in MC should be based not only upon clinical features and the inheritance pattern but also on molecular genetic analysis and ideally functional expression data.

Andersen-Tawil syndrome: new potassium channel mutations and possible phenotypic variation.

OBJECTIVE: To evaluate clinical, genetic, and electrophysiologic features of patients with Andersen-Tawil syndrome (ATS) in the United Kingdom. METHODS: Clinical and neurophysiologic evaluation was conducted of 11 families suspected to have ATS. Molecular genetic analysis of each proband was performed by direct DNA sequencing of the entire coding region of KCNJ2. Control samples were screened by direct DNA sequencing. The electrophysiologic consequences of several new mutations were studied in an oocyte expression system. RESULTS: All 11 ATS families harbored pathogenic mutations in KCNJ2 with six mutations not previously reported. Some unusual clinical features including renal tubular defect, CNS involvement, and dental and phonation abnormalities were observed. Five mutations (T75M, D78G, R82Q, L217P, and G300D) were expressed, all of which resulted in nonfunctional channels when expressed alone, and co-expression with wild-type (WT) KCNJ2 demonstrated a dominant negative effect. CONCLUSION: Six new disease-causing mutations in KCNJ2 were identified, one of which was in a PIP2 binding site. Molecular expression studies indicated that five of the mutations exerted a dominant negative effect on the wild-type allele. KCNJ2 mutations are an important cause of ATS in the UK.

Late-onset episodic ataxia type 2 due to an in-frame insertion in CACNA1A.

Episodic ataxia type 2 (EA2) is caused by calcium channel (CACNA1A) mutations and typically begins before age 20 years. The molecular basis of late-onset EA2 is unclear. The authors describe a case of late-onset EA2 associated with the first multiple-base pair insertion in CACNA1A. Molecular expression revealed evidence of impaired calcium channel function, suggesting that genetically induced reduction in calcium channel function may associate with cases of late-onset EA2.

Neurological phenotype and synaptic function in mice lacking the CaV1.3 alpha subunit of neuronal L-type voltage-dependent Ca2+ channels.

Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the CaV1.3 (alpha 1D) alpha subunit of L-type Ca2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. CaV1.3 alpha 1 subunit knockout (CaV1.3 alpha 1(-/-)) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. CaV1.3 alpha 1(-/-) mice were also examined for differences in N-methyl-D-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 microM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[3H]isradipine binding sites in brain homogenates was reduced by 20-25% in CaV1.3 alpha 1(-/-) mice, without any detectable change in CaV1.2 (alpha 1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of CaV1.3 alpha 1 subunit expression there is sufficient residual activity of other Ca2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.

Outcome of ventilatory support for acute respiratory failure in motor neurone disease.

OBJECTIVES: To review the outcome of acute ventilatory support in patients presenting acutely with respiratory failure, either with an established diagnosis of motor neurone disease (MND) or with a clinical event where the diagnosis of MND has not yet been established. METHODS: Outcome was reviewed in 24 patients with respiratory failure due to MND who received endotracheal intubation and intermittent positive pressure ventilation either at presentation or as a result of the unexpected development of respiratory failure. Patients presenting to local hospitals with acute respiratory insufficiency and requiring tracheal intubation, ventilatory support, and admission to an intensive therapy unit (ITU) before transfer to a regional respiratory care unit were selected. Clinical features of presentation, management, and outcome were studied. RESULTS: 24 patients with MND were identified, all being intubated and ventilated acutely within hours of presentation. 17 patients (71%) were admitted in respiratory failure before the diagnosis of MND had been made; the remaining seven patients (29%) were already known to have MND but deteriorated rapidly such that intubation and ventilation were initiated acutely. Seven patients (29%) died on ITU (between seven and 54 days after admission). 17 patients (71%) were discharged from ITU. 16 patients (67%) received long term respiratory support and one patient required no respiratory support following tracheal extubation. The daily duration of support that was required increased gradually with time. CONCLUSION: When a patient with MND is ventilated acutely, with or without an established diagnosis, independence from the ventilator is rarely achieved. Almost all of these patients need long term ventilatory support and the degree of respiratory support increases with time as the disease progresses. The aim of management should be weaning the patient to the minimum support compatible with symptomatic relief and comfort. Respiratory failure should be anticipated in patients with MND when the diagnosis has been established.