modCHIMERA: a novel murine closed-head model of moderate traumatic brain injury.

Traumatic brain injury is a major source of global disability and mortality. Preclinical TBI models are a crucial component of therapeutic investigation. We report a tunable, monitored model of murine non-surgical, diffuse closed-head injury-modCHIMERA-characterized by impact as well as linear and rotational acceleration. modCHIMERA is based on the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) platform. We tested this model at 2 energy levels: 1.7 and 2.1 Joules-substantially higher than previously reported for this system. Kinematic analysis demonstrated linear acceleration exceeding injury thresholds in humans, although outcome metrics tracked impact energy more closely than kinematic parameters. Acute severity metrics were consistent with a complicated-mild or moderate TBI, a clinical population characterized by high morbidity but potentially reversible pathology. Axonal injury was multifocal and bilateral, neuronal death was detected in the hippocampus, and microglial neuroinflammation was prominent. Acute functional analysis revealed prolonged post-injury unconsciousness, and decreased spontaneous behavior and stimulated neurological scores. Neurobehavioral deficits were demonstrated in spatial learning/memory and socialization at 1-month. The overall injury profile of modCHIMERA corresponds with the range responsible for a substantial portion of TBI-related disability in humans. modCHIMERA should provide a reliable platform for efficient analysis of TBI pathophysiology and testing of treatment modalities.

Neurofilament light chain levels in ventricular cerebrospinal fluid after acute aneurysmal subarachnoid haemorrhage.

PURPOSE: The contribution of axonal injury to brain damage after aneurysmal subarachnoid haemorrhage (aSAH) is unknown. Neurofilament light chain (NF-L), a component of the axonal cytoskeleton, has been shown to be elevated in the cerebrospinal fluid of patients with many types of axonal injury. We hypothesised that patients with aSAH would have elevated cerebrospinal fluid (CSF) NF-L levels and sought to explore the clinical correlates of CSF NF-L dynamics. METHODS: Serial ventricular CSF (vCSF) samples were collected from 35 patients with aSAH for up to 15 days. vCSF NF-L measurements were determined by enzyme-linked immunosorbent assay. NF-L levels were analysed in relation to acute clinical status, radiological findings and 6-month outcomes. RESULTS: vCSF NF-L concentrations were elevated in all patients with aSAH. Patients with early cerebral ischaemia (ECI), defined as a CT hypodense lesion visible within the first 3 days, had higher acute vCSF NF-L levels than patients without ECI. These elevated NF-L levels were similar in patients with ECI associated with intracranial haemorrhage and ECI associated with surgical/endovascular complications. vCSF NF-L levels did not differ as a function of acute clinical status, clinical vasospasm, delayed cerebral ischaemia or 6-month Glasgow Outcome Scale. CONCLUSIONS: Elevated vCSF NF-L levels may in part reflect increased injury to axons associated with ECI. However, our results suggest that axonal injury after aSAH as reflected by release of NF-L into the CSF may not play a major role in either secondary adverse events or long-term clinical outcomes.

Detection of traumatic axonal injury with diffusion tensor imaging in a mouse model of traumatic brain injury.

Traumatic axonal injury (TAI) is thought to be a major contributor to cognitive dysfunction following traumatic brain injury (TBI), however TAI is difficult to diagnose or characterize non-invasively. Diffusion tensor imaging (DTI) has shown promise in detecting TAI, but direct comparison to histologically-confirmed axonal injury has not been performed. In the current study, mice were imaged with DTI, subjected to a moderate cortical controlled impact injury, and re-imaged 4-6 h and 24 h post-injury. Axonal injury was detected by amyloid beta precursor protein (APP) and neurofilament immunohistochemistry in pericontusional white matter tracts. The severity of axonal injury was quantified using stereological methods from APP stained histological sections. Two DTI parameters--axial diffusivity and relative anisotropy--were significantly reduced in the injured, pericontusional corpus callosum and external capsule, while no significant changes were seen with conventional MRI in these regions. The contusion was easily detectable on all MRI sequences. Significant correlations were found between changes in relative anisotropy and the density of APP stained axons across mice and across subregions spanning the spatial gradient of injury. The predictive value of DTI was tested using a region with DTI changes (hippocampal commissure) and a region without DTI changes (anterior commissure). Consistent with DTI predictions, there was histological detection of axonal injury in the hippocampal commissure and none in the anterior commissure. These results demonstrate that DTI is able to detect axonal injury, and support the hypothesis that DTI may be more sensitive than conventional imaging methods for this purpose.

G-protein inhibition of N- and P/Q-type calcium channels: distinctive elementary mechanisms and their functional impact.

Voltage-dependent G-protein inhibition of presynaptic Ca(2+) channels is a key mechanism for regulating synaptic efficacy. G-protein betagamma subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as "willing" and "reluctant" modes of gating. Recent macroscopic electrophysiological data hint that only N-type, but not P/Q-type channels can open in the reluctant mode, a distinction that could enrich the dimensions of synaptic modulation arising from channel inhibition. Here, using high-resolution single-channel recording of recombinant channels, we directly distinguished this core contrast in the prevalence of reluctant openings. Single, inhibited N-type channels manifested relatively infrequent openings of submillisecond duration (reluctant openings), which differed sharply from the high-frequency, millisecond gating events characteristic of uninhibited channels. By contrast, inhibited P/Q-type channels were electrically silent at the single-channel level. The functional impact of the differing inhibitory mechanisms was revealed in macroscopic Ca(2+) currents evoked with neuronal action potential waveforms (APWs). Fitting with a change in the manner of opening, inhibition of such N-type currents produced both decreased current amplitude and temporally advanced waveform, effects that would not only reduce synaptic efficacy, but also influence the timing of synaptic transmission. On the other hand, inhibition of P/Q-type currents evoked by APWs showed diminished amplitude without shape alteration, as expected from a simple reduction in the number of functional channels. Variable expression of N- and P/Q-type channels at spatially distinct synapses therefore offers the potential for custom regulation of both synaptic efficacy and synchrony, by G-protein inhibition.

Release-independent short-term synaptic depression in cultured hippocampal neurons.

Short-term synaptic plasticity may dramatically influence neuronal information transfer, yet the underlying mechanisms remain incompletely understood. In autapses (self-synapses) formed by cultured hippocampal neurons, short-term synaptic depression (STD) had several unusual features. (1) Reduction of neurotransmitter release probability with Cd(2+), a blocker of voltage-gated calcium channels, did not change depression. (2) Lowering [Ca(2+)](o) and/or raising [Mg(2+)](o) had little effect on STD in cells with strong baseline depression, but in cells with more modest baseline depression, it reduced the depression. (3) Random variations in the size of initial EPSCs did not influence successive EPSC sizes. These findings were inconsistent with release-dependent mechanisms, such as vesicle depletion, post-synaptic receptor desensitization, and autoreceptor inhibition. Instead, other results suggested that changes in action potentials (APs) contributed to depression. The somatic APs declined in amplitude with repetitive stimulation, and modest reduction of AP amplitudes with tetrodotoxin inhibited EPSCs. Notably, tetrodotoxin also increased depression. Similar changes in axonal APs could produce STD in at least two ways. First, decreasing presynaptic spike amplitudes could reduce calcium entry and release probability. Alternatively, APs could fail to propagate through some axonal branches, reducing the number of active synapses. To explore these possibilities, we derived the expected variance of EPSCs for the two scenarios. Experimentally, the variance increased and then decreased on average with successive responses during trains of APs, confirming a unique prediction from the conduction failure scenario. Thus, STD had surprising properties, incompatible with commonly postulated mechanisms but consistent with AP conduction failure at axonal branches.

Relief of G-protein inhibition of calcium channels and short-term synaptic facilitation in cultured hippocampal neurons.

G-protein inhibition of voltage-gated calcium channels can be transiently relieved by repetitive physiological stimuli. Here, we provide evidence that such relief of inhibition contributes to short-term synaptic plasticity in microisland-cultured hippocampal neurons. With G-protein inhibition induced by the GABA(B) receptor agonist baclofen or the adenosine A1 receptor agonist 2-chloroadenosine, short-term synaptic facilitation emerged during action potential trains. The facilitation decayed with a time constant of approximately 100 msec. However, addition of the calcium channel inhibitor Cd(2+) at 2-3 microM had no such effect and did not alter baseline synaptic depression. As expected of facilitation from relief of channel inhibition, analysis of miniature EPSCs implicated presynaptic modulation, and elevating presynaptic Ca(2+) entry blunted the facilitation. Most telling was the near occlusion of synaptic facilitation after selective blockade of P/Q- but not N-type calcium channels. This was as predicted from experiments using recombinant calcium channels expressed in human embryonic kidney (HEK) 293 cells; we found significantly stronger relief of G-protein inhibition in recombinant P/Q- versus N-type channels during action potential trains. G-protein inhibition in HEK 293 cells was induced via recombinant M2 muscarinic acetylcholine receptors activated by carbachol, an acetylcholine analog. Thus, relief of G-protein inhibition appears to produce a novel form of short-term synaptic facilitation in cultured neurons. Similar short-term synaptic plasticity may be present at a wide variety of synapses, as it could occur during autoreceptor inhibition by glutamate or GABA, heterosynaptic inhibition by GABA, tonic adenosine inhibition, and in many other instances.

Preferential closed-state inactivation of neuronal calcium channels.

We have investigated the inactivation mechanism of neuronal N-, P/Q-, and R-type calcium channels. Although channels inactivate slowly during square-pulse depolarization, as observed previously, we now find that they inactivate profoundly during a train of action potential (AP) waveforms. The apparent paradox arises from a voltage-dependent mechanism in which channels inactivate preferentially from intermediate closed states along the activation pathway. Inactivation can therefore extend beyond the brief duration of AP waveforms to continue between spikes, as the channel undergoes repetitive cycles of activation and deactivation. The extent of inactivation during a train is strongly affected by the subunit composition of channels. Preferential closed-state inactivation of neuronal calcium channels could produce widely variable depression of Ca2+ entry during a train of APs.

Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells.

1. A variety of neurotransmitters act through G-protein-coupled receptors to decrease synaptic transmission, largely by inhibiting the voltage-gated calcium channels that trigger neurotransmitter release. However, these presynaptic calcium channels are typically inaccessible to electrophysiological characterization. We have reconstituted a part of this inhibition using recombinant P/Q-type calcium channels and M2 acetylcholine receptors in HEK 293 cells. 2. One of the most interesting features of G-protein inhibition of calcium channels is that strong step depolarization transiently relieves the inhibition. We have found that short bursts of action potential voltage waveforms can also relieve the inhibition, increasing calcium current through G-protein-inhibited channels but not through uninhibited channels. 3. The extent of this relief increased linearly with the duration of the action potential waveforms. 4. This result provides the strongest evidence to date favouring the possibility that relief of G-protein inhibition can occur during high frequency trains of action potentials. This effect may constitute a novel form of short-term synaptic plasticity that is sensitive to action potential timing and duration.