Claire A. Baker

Nitric Oxide Is a Volume Transmitter Regulating Postsynaptic Excitability at a Glutamatergic Synapse

Neuronal nitric oxide synthase (nNOS) is broadly expressed in the brain and associated with synaptic plasticity through NMDAR-mediated calcium influx. However, its physiological activation and the mechanisms by which nitric oxide (NO) influences synaptic transmission have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held to characterize NO modulation at this glutamatergic synapse in the auditory pathway. NO is generated in an activity-dependent manner by MNTB principal neurons receiving a calyceal synaptic input. It acts in the target neuron and adjacent inactive neurons to modulate excitability and synaptic efficacy, inhibiting postsynaptic Kv3 potassium currents (via phosphorylation), reducing EPSCs and so increasing action potential duration and reducing transmission fidelity. We conclude that NO serves as a volume transmitter and slow dynamic modulator, integrating spontaneous and evoked neuronal firing, thereby providing an index of global activity and regulating information transmission across a population of active and inactive neurons.

Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K+ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half‐activated at −9.2 ± 2.1 mV and half‐inactivated at −35.9 ± 1.5 mV. It is blocked by several non‐specific potassium channel antagonists including quinine (100 μm) and high concentrations of extracellular tetraethylammonium (TEA; IC50= 11.8 mm), but no specific antagonists were found. These characteristics are similar to recombinant Kv2‐mediated currents. Quantitative RT‐PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage‐gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage‐gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current‐clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2‐containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing.

Prions Are Secreted into the Oral Cavity in Sheep with Preclinical Scrapie

A major concern in prion disease transmission is the spread of the disease agent by means of secretions and excretions. We analyzed buccal swab samples obtained from preclinical scrapie-infected sheep by concentrating the collected prions on silicon dioxide, followed by amplification by serial protein misfolding cyclic amplification. Data clearly demonstrate that prions are present in buccal swab samples from sheep with a VRQ/VRQ PRNP genotype during preclinical scrapie infection. These data describe for the first time to our knowledge the secretion of prions into the oral cavity of sheep, a finding with implications for the transmission of ovine scrapie and very likely other prion diseases.

Environmental Sources of Scrapie Prions

ABSTRACT Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.

Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep

ABSTRACT The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrPSc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.

Detection of prions in the faeces of sheep naturally infected with classical scrapie

Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.

The Oral Secretion of Infectious Scrapie Prions Occurs in Preclinical Sheep with a Range of PRNP Genotypes

ABSTRACT Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrPSc within lymphoreticular tissues. PrPSc was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.

Kv4 (A-type) potassium currents in the mouse medial nucleus of the trapezoid body.

Principal neurones of the mouse medial nucleus of the trapezoid body (MNTB) possess multiple voltage‐gated potassium currents, including a transient outward current (or A‐current), which is characterized here. The A‐current exhibited rapid voltage‐dependent inactivation and was half inactivated at resting membrane potentials. Following a hyperpolarizing pre‐pulse to remove inactivation, the peak transient current was 1.07 nA at −17 mV. The pharmacological characteristics of this A‐current were consistent with Kv4 subunits in expression studies; the A‐current was resistant to block by tetraethylammonium and dendrotoxin‐I but sensitive to millimolar concentrations of 4‐aminopyridine and 5 µm hanatoxin. Immunohistochemistry confirmed that Kv4.3 sub‐units are present in the MNTB. In a single‐compartment model of an MNTB neurone, the A‐current served to accelerate the decay of the initial action potentials in a stimulus train and suggested that removal of A‐current steady‐state inactivation could raise firing threshold for non‐calyceal synaptic inputs. This A‐type current was not observed in the rat.

In vitro amplification of prions from milk in the detection of subclinical infections

Prions can be amplified by serial protein misfolding cyclic amplification (sPMCA) from the milk of a high proportion of apparently healthy, scrapie exposed sheep with PRNP genotypes not previously associated with high disease penetrance1. These data strongly suggest the widespread presence of subclinical scrapie infections within scrapie-exposed flocks containing sheep with a range of susceptible PRNP genotypes. These data also lead to the hypothesis that similar subclinical disease states may be common for other animal and human prion diseases. Furthermore, the application of sPMCA to milk provides a method to detect such subclinical disease. Here, we describe the high level amplification of bovine spongiform encephalopathy (BSE) prions from both ovine and bovine origin, a methodology that will facilitate the detection of any prions secreted within bovine and ovine milk during subclinical and clinical BSE disease.

Circulation of prions within dust on a scrapie affected farm

Prion diseases are fatal neurological disorders that affect humans and animals. Scrapie of sheep/goats and Chronic Wasting Disease (CWD) of deer/elk are contagious prion diseases where environmental reservoirs have a direct link to the transmission of disease. Using protein misfolding cyclic amplification we demonstrate that scrapie PrPSc can be detected within circulating dusts that are present on a farm that is naturally contaminated with sheep scrapie. The presence of infectious scrapie within airborne dusts may represent a possible route of infection and illustrates the difficulties that may be associated with the effective decontamination of such scrapie affected premises.