Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

A Balanced Approach for Cannabidiol Use in Chronic Pain.

Cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa L., has gained traction as a potential treatment for intractable chronic pain in many conditions. Clinical evidence suggests that CBD provides therapeutic benefit in certain forms of epilepsy and imparts analgesia in certain conditions, and improves quality of life. CBD continues to be Schedule I or V on the list of controlled substances of the Drug Enforcement Agency of the United States. However, preparations labeled CBD are available publicly in stores and on the streets. However, use of CBD does not always resolve pain. CBD purchased freely entails the risk of adulteration by potentially hazardous chemicals. As well, CBD use by pregnant women is rising and poses a major health-hazard for future generations. In this mini-review, we present balanced and unbiased pre-clinical and clinical findings for the beneficial effects of CBD treatment on chronic pain and its deleterious effects on prenatal development.

Enhanced Activation of HCN Channels Reduces Excitability and Spike-Timing Regularity in Maturing Vestibular Afferent Neurons.

Vestibular ganglion neurons (VGNs) transmit information along parallel neuronal pathways whose signature distinction is variability in spike-timing; some fire at regular intervals while others fire at irregular intervals. The mechanisms driving timing differences are not fully understood but two opposing (but not mutually exclusive) hypotheses have emerged. In the first, regular-spiking is inversely correlated to the density of low-voltage-gated potassium currents (IKL). In the second, regular spiking is directly correlated to the density of hyperpolarization-activated cyclic nucleotide-sensitive currents (IH). Supporting the idea that variations in ion channel composition shape spike-timing, VGNs from the first postnatal week respond to synaptic-noise-like current injections with irregular-firing patterns if they have IKL and with more regular firing patterns if they do not. However, in vitro firing patterns are not as regular as those in vivo Here we considered whether highly-regular spiking requires IH currents and whether this dependence emerges later in development after channel expression matures. We recorded from rat VGN somata of either sex aged postnatal day (P)9-P21. Counter to expectation, in vitro firing patterns were less diverse, more transient-spiking, and more irregular at older ages than at younger ages. Resting potentials hyperpolarized and resting conductance increased, consistent with developmental upregulation of IKL Activation of IH (by increasing intracellular cAMP) increased spike rates but not spike-timing regularity. In a model, we found that activating IH counter-intuitively suppressed regularity by recruiting IKL Developmental upregulation in IKL appears to overwhelm IH These results counter previous hypotheses about how IH shapes vestibular afferent responses.SIGNIFICANCE STATEMENT Vestibular sensory information is conveyed on parallel neuronal pathways with irregularly-firing neurons encoding information using a temporal code and regularly-firing neurons using a rate code. This is a striking example of spike-timing statistics influencing information coding. Previous studies from immature vestibular ganglion neurons (VGNs) identified hyperpolarization-activated mixed cationic currents (IH) as driving highly-regular spiking and proposed that this influence grows with the current during maturation. We found that IH becomes less influential, likely because maturing VGNs also acquire low-voltage-gated potassium currents (IKL), whose inhibitory influence opposes IH Because efferent activity can partly close IKL, VGN firing patterns may become more receptive to extrinsic control. Spike-timing regularity likely relies on dynamic ion channel properties and complementary specializations in synaptic connectivity.

Therapeutic modulation of cerebral microhemorrhage in a mouse model of cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: The aging brain demonstrates frequent MRI and pathological evidence of cerebral microbleeds, which are often associated with cerebral amyloid angiopathy. To develop new therapeutic strategies for this disorder, we studied cerebral microhemorrhage in a well-characterized mouse model of cerebral amyloid angiopathy. METHODS: Tg2576 mice were studied at ages ranging from 2 to 21 months. Spontaneous and induced microscopic bleeding was analyzed with and without a passive anti-amyloid immunization regimen and dietary supplementation of ischemic stroke prevention medication dipyridamole. RESULTS: Areas of microhemorrhage were easily demonstrated and were significantly more prominent in the oldest mice and in animals treated with anti-amyloid immunotherapy. Dipyridamole supplementation in the diet generated plasma levels >790 ng/mL within the range seen clinically. Dipyridamole treatment did not worsen frequency and size of cerebral microscopic bleeding. CONCLUSIONS: The Tg2576 mouse is a useful model to study progression and modification of spontaneous and immunotherapy-induced cerebral microhemorrhage. Absence of microhemorrhage worsening with dipyridamole treatment suggests a potential therapeutic role of this agent when ischemic and microhemorrhagic lesions coexist.