Inhibition of L-Type Ca 2+ Channels by TRPC1-STIM1 Complex Is Essential for the Protection of Dopaminergic Neurons

Loss of dopaminergic (DA) neurons leads to Parkinson’s disease; however, the mechanism(s) for the vulnerability of DA neurons is(are) not fully understood. We demonstrate that TRPC1 regulates the L-type Ca2 channel that contributes to the rhythmic activity of adult DA neurons in the substantia nigra region. Store depletion that activates TRPC1, via STIM1, inhibits the frequency and amplitude of the rhythmic activity in DA neurons of wild-type, but not in TRPC1/, mice. Similarly, TRPC1/ substantia nigra neurons showed increased L-type Ca2 currents, decreased stimulation-dependent STIM1-Cav1.3 interaction, and decreased DA neurons. L-type Ca2 currents and the open channel probability of Cav1.3 channels were also reduced upon TRPC1 activation, whereas increased Cav1.3 currents were observed upon STIM1 or TRPC1 silencing. Increased interaction between Cav1.3-TRPC1-STIM1 was observed upon store depletion and the loss of either TRPC1 or STIM1 led to DA cell death, which was prevented by inhibiting L-type Ca2 channels. Neurotoxins that mimic Parkinson’s disease increased Cav1.3 function, decreased TRPC1 expression, inhibited Tg-mediated STIM1-Cav1.3 interaction, and induced caspase activation. Importantly, restoration of TRPC1 expression not only inhibited Cav1.3 function but increased cell survival. Together, we provide evidence that TRPC1 suppresses Cav1.3 activity by providing an STIM1-based scaffold, which is essential for DA neuron survival.Fil: Sun, Yuyang. University of North Dakota; Estados UnidosFil: Zhang, Haopeng. University of North Dakota; Estados UnidosFil: Selvaraj, Senthil. University of North Dakota; Estados UnidosFil: Sukumaran, Pramod. University of North Dakota; Estados UnidosFil: Lei, Saobo. University of North Dakota; Estados UnidosFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institutes of Environmental Health Sciences; Estados UnidosFil: Singh, Brij B.. University of North Dakota; Estados Unido

Corticotropin-releasing factor facilitates epileptiform activity in the entorhinal cortex: roles of CRF2 receptors and PKA pathway.

Whereas corticotropin-releasing factor (CRF) has been considered as the most potent epileptogenic neuropeptide in the brain, its action site and underlying mechanisms in epilepsy have not been determined. Here, we found that the entorhinal cortex (EC) expresses high level of CRF and CRF2 receptors without expression of CRF1 receptors. Bath application of CRF concentration-dependently increased the frequency of picrotoxin (PTX)-induced epileptiform activity recorded from layer III of the EC in entorhinal slices although CRF alone did not elicit epileptiform activity. CRF facilitated the induction of epileptiform activity in the presence of subthreshold concentration of PTX which normally would not elicit epileptiform activity. Bath application of the inhibitor for CRF-binding proteins, CRF6-33, also increased the frequency of PTX-induced epileptiform activity suggesting that endogenously released CRF is involved in epileptogenesis. CRF-induced facilitation of epileptiform activity was mediated via CRF2 receptors because pharmacological antagonism and knockout of CRF2 receptors blocked the facilitatory effects of CRF on epileptiform activity. Application of the adenylyl cyclase (AC) inhibitors blocked CRF-induced facilitation of epileptiform activity and elevation of intracellular cyclic AMP (cAMP) level by application of the AC activators or phosphodiesterase inhibitor increased the frequency of PTX-induced epileptiform activity, demonstrating that CRF-induced increases in epileptiform activity are mediated by an increase in intracellular cAMP. However, application of selective protein kinase A (PKA) inhibitors reduced, not completely blocked CRF-induced enhancement of epileptiform activity suggesting that PKA is only partially required. Our results provide a novel cellular and molecular mechanism whereby CRF modulates epilepsy

A protocol for preparation and transfection of rat entorhinal cortex organotypic cultures for electrophysiological whole-cell recordings

Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC) is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG)-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of “bullets” carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations

The entorhinal neurons expresses CRF and CRF₂ receptors but not CRF₁ receptors.

<p><b>A</b>, Immunoreactivity for CRF (<i>upper</i>) and detection of CRF by western blot (<i>lower</i>). <i>Upper right</i>: high magnification of the region marked in the left. <b>B</b>, Lack of immunoreactivity (<i>upper</i>) and protein band (<i>lower</i>) for CRF₁ receptors. <b>C</b>, The entorhinal neurons showed immunoreactivity (<i>upper</i>) for CRF₂ receptors and western blot detected a band close to the molecular mass of CRF₂ receptors in the lysates of the EC (<i>lower</i>).</p

CRF facilitates epileptiform activity via activation of CRF₂ receptors.

<p><b>A</b>, Pretreatment of slices with and continuous bath application of K41498, a selective CRF₂ antagonist, blocked CRF-mediated increases in epileptiform activity. <b>B</b>, Pretreatment of slices with and continuous bath application of astressin 2B, another selective CRF₂ antagonist, blocked CRF-mediated increases in epileptiform activity. <b>C</b>, Pretreatment of slices with and continuous bath application of NBI 27914, a selective CRF₁ antagonist, failed to alter significantly CRF-mediated increases in epileptiform activity. <b>D</b>, Pretreatment of slices with and continuous bath application of CP 154526, another selective CRF₁ antagonist, did not change the facilitatory effect of CRF on epileptiform activity. <b>E</b>, Application of CRF increased epileptiform activity in WT mice. <b>F</b>, Application of CRF did not induce an increase in epileptiform activity in CRF₂ KO mice.</p