scholarly journals Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
P. Kleis ◽  
E. Paschen ◽  
U. Häussler ◽  
Y. A. Bernal Sierra ◽  
C. A. Haas
Keyword(s):  
Side Effects ◽  
Potassium Channel ◽  
Neuronal Activity ◽  
Mammalian Cells ◽  
Viral Vector ◽  
Epileptiform Activity ◽  
Field Potential ◽  
Pyramidal Cells

Abstract Background Optogenetic tools allow precise manipulation of neuronal activity via genetically encoded light-sensitive proteins. Currently available optogenetic inhibitors are not suitable for prolonged use due to short-lasting photocurrents, tissue heating, and unintended changes in ion distributions, which may interfere with normal neuron physiology. To overcome these limitations, a novel potassium channel-based optogenetic silencer, named PACK, was recently developed. The PACK tool has two components: a photoactivated adenylyl cyclase from Beggiatoa (bPAC) and a cAMP-dependent potassium channel, SthK, which carries a large, long-lasting potassium current in mammalian cells. Previously, it has been shown that activating the PACK silencer with short light pulses led to a significant reduction of neuronal firing in various in vitro and acute in vivo settings. Here, we examined the viability of performing long-term studies in vivo by looking at the inhibitory action and side effects of PACK and its components in healthy and epileptic adult male mice. Results We targeted hippocampal cornu ammonis (CA1) pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-min intermittent (0.1 Hz) illumination, especially in the gamma frequency range. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Light activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. Furthermore, we applied bPAC and PACK in the contralateral hippocampus of chronically epileptic mice following a unilateral injection of intrahippocampal kainate. Unexpectedly, the expression of bPAC in the contralateral CA1 area was sufficient to prevent the spread of spontaneous epileptiform activity from the seizure focus to the contralateral hippocampus. Conclusion Our study highlights the PACK tool as a potent optogenetic inhibitor in vivo. However, further refinement of its light-sensitive domain is required to avoid unexpected physiological changes.

scholarly journals In vivo characterization and application of a novel potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

2021 ◽  
Author(s):  
Piret Kleis ◽  
Enya Paschen ◽  
Ute Haeussler ◽  
Yinth Andrea Bernal Sierra ◽  
Carola A. Haas
Keyword(s):  
Side Effects ◽  
Potassium Channel ◽  
Neuronal Activity ◽  
Viral Vector ◽  
Epileptiform Activity ◽  
Field Potential ◽  
Pyramidal Cells ◽  
Light Sensitivity

The performance of available optogenetic inhibitors remains insufficient due to low light sensitivity, short-lasting photocurrents, and unintended changes in ion distributions. To overcome these limitations, a novel potassium channel-based optogenetic silencer was developed and successfully applied in various in vitro and acute in vivo settings (Bernal Sierra et al., 2018). This tool, a two-component construct called PACK, comprises a photoactivated adenylyl cyclase (bPAC) and a cAMP-dependent potassium channel (SthK). Here, we examined the long-term inhibitory action and side effects of the PACK construct in healthy and epileptic adult male mice. We targeted hippocampal CA1 pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from the CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-minute intermittent illumination, especially in the beta and gamma frequency ranges. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Additionally, light-activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. In chronically epileptic mice, the dark activity of bPAC/PACK in CA1 prevented the spread of spontaneous epileptiform activity from the seizure focus to the contralateral bPAC/PACK-expressing hippocampus. Taken together, the PACK tool is a potent optogenetic inhibitor but requires refinement of its light-sensitive domain to avoid unexpected physiological changes.

Enhanced Stability and Controlled Delivery of MOF Encapsulated Vaccines and Their Immunogenic Response In Vivo

2018 ◽  
Author(s):  
Michael Luzuriaga ◽  
Raymond P. Welch ◽  
Madushani Dharmawardana ◽  
Candace Benjamin ◽  
Shaobo Li ◽  
...  
Keyword(s):  
Viral Vector ◽  
Controlled Delivery ◽  
Conformational Structure ◽  
Spectroscopy Analysis ◽  
Zeolitic Imidazolate Framework ◽  
Structural Conformation ◽  
Depth Study ◽  
Viral Nanoparticle

<div><div><div><p>Vaccines have an innate tendency to lose their structural conformation upon environmental and chemical stressors. A loss in conformation reduces the therapeutic ability to prevent the spread of a pathogen. Herein, we report an in-depth study of zeolitic imidazolate framework-8 (ZIF-8) and its ability to provide protection for a model viral vector against dena- turing conditions. The immunoassay and spectroscopy analysis together demonstrate enhanced thermal and chemical stability to the conformational structure of the encapsulated viral nanoparticle. The long-term biological activity of this virus-ZIF composite was investigated in animal models to further elucidate the integrity of the encapsulated virus, the bio-safety, and immunogenicity of the overall composite. Additionally, histological analysis found no observable tissue damage in the skin or vital organs in mice, following multiple subcutaneous administrations. This study shows that ZIF-based protein composites are strong candidates for improved preservation of proteinaceous drugs, are biocompatible, and capable of controlling the release and adsorption of drugs in vivo.</p></div></div></div>

scholarly journals Aη-α and Aη-β peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Maria Mensch ◽  
Jade Dunot ◽  
Sandy M. Yishan ◽  
Samuel S. Harris ◽  
Aline Blistein ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Ex Vivo ◽  
Long Term Potentiation ◽  
Network Activity ◽  
Strongly Correlated ◽  
App Processing ◽  
Term Potentiation ◽  
Hippocampal Network

Abstract Background Amyloid precursor protein (APP) processing is central to Alzheimer’s disease (AD) etiology. As early cognitive alterations in AD are strongly correlated to abnormal information processing due to increasing synaptic impairment, it is crucial to characterize how peptides generated through APP cleavage modulate synapse function. We previously described a novel APP processing pathway producing η-secretase-derived peptides (Aη) and revealed that Aη–α, the longest form of Aη produced by η-secretase and α-secretase cleavage, impaired hippocampal long-term potentiation (LTP) ex vivo and neuronal activity in vivo. Methods With the intention of going beyond this initial observation, we performed a comprehensive analysis to further characterize the effects of both Aη-α and the shorter Aη-β peptide on hippocampus function using ex vivo field electrophysiology, in vivo multiphoton calcium imaging, and in vivo electrophysiology. Results We demonstrate that both synthetic peptides acutely impair LTP at low nanomolar concentrations ex vivo and reveal the N-terminus to be a primary site of activity. We further show that Aη-β, like Aη–α, inhibits neuronal activity in vivo and provide confirmation of LTP impairment by Aη–α in vivo. Conclusions These results provide novel insights into the functional role of the recently discovered η-secretase-derived products and suggest that Aη peptides represent important, pathophysiologically relevant, modulators of hippocampal network activity, with profound implications for APP-targeting therapeutic strategies in AD.

Nonsynaptic epileptogenesis in the mammalian hippocampus in vitro. I. Development of seizurelike activity in low extracellular calcium

1986 ◽  
Vol 56 (2) ◽  
pp. 409-423 ◽  
Author(s):  
A. Konnerth ◽  
U. Heinemann ◽  
Y. Yaari
Keyword(s):  
Epileptiform Activity ◽  
Large Population ◽  
Hippocampal Slices ◽  
Field Potential ◽  
Pyramidal Cells ◽  
Discharge Zone ◽  
Critical Threshold ◽  
Mean Velocity ◽  
Stratum Pyramidale ◽  
Ca1 Area

Epileptiform activity induced in rat hippocampal slices by lowering extracellular Ca2+ concentration ([Ca2+]o) was studied with extracellular and intracellular recordings. Perfusing the slices with low Ca2+ (less than or equal to 0.2 mM) or EGTA-containing solutions blocked the synaptic responses of hippocampal pyramidal cells (HPCs). Despite the block, spontaneous paroxysms, termed seizurelike events (SLEs), appeared in the CA1 area and then recurred regularly at a stable frequency. Transient hypoxia accelerated their development and increased their frequency. When [Ca2+]o was raised in a stepwise manner, the SLEs disappeared at 0.3 mM. With extracellular recording from the CA1 stratum pyramidale, a SLE was characterized by a large negative shift in the field potential, which lasted for several seconds. During this period a large population of CA1 neurons discharged intensely and often in synchrony, as concluded from the frequent appearance of population spikes. Synchronization, however, was not a necessary precursor for the development of paroxysmal activity, but seemed to be the end result of massive neuronal excitation. The cellular counterpart of a SLE, as revealed by intracellular recording from HPCs in the discharge zone of the paroxysms, was a long-lasting depolarization shift (LDS) of up to 20 mV. This was accompanied by accelerated firing of the neuron. A prolonged after-hyperpolarization succeeded each LDS and arrested cell firing. Brief (approximately 50 ms) bursts were commonly observed before LDS onset. Single electrical stimuli applied focally to the stratum pyramidale or alveus evoked paroxysms identical to the spontaneous SLEs, provided they surpassed a critical threshold intensity. Subthreshold stimuli elicited only small local responses, whereas stimuli of varied suprathreshold intensities evoked the same maximal SLEs. Thus the buildup of a SLE is an all or nothing or a regenerative process, which mobilizes the majority, if not all, of the local neuronal population. Each SLE was followed by absolute and relative refractory periods during which focal stimulation was, respectively, ineffective and less effective in evoking a maximal SLE. In most slices the spontaneous SLEs commenced at a "focus" located in the CA1a subarea (near the subiculum). SLEs evoked by focal stimulation arose near the stimulating electrode. From their site of origin the paroxysmal discharges spread transversely through the entire CA1 area at a mean velocity of 1.74 mm/s. Consequently, the discharge zone of a SLE could encompass for several seconds the entire CA1 area.(ABSTRACT TRUNCATED AT 400 WORDS)

Epileptiform activity induced by low chloride medium in the CA1 subfield of the hippocampal slice

1990 ◽  
Vol 64 (6) ◽  
pp. 1747-1757 ◽  
Author(s):  
M. Avoli ◽  
C. Drapeau ◽  
P. Perreault ◽  
J. Louvel ◽  
R. Pumain
Keyword(s):  
Membrane Potential ◽  
Large Amplitude ◽  
Action Potentials ◽  
Hippocampal Slice ◽  
Epileptiform Activity ◽  
Field Potential ◽  
Pyramidal Cells ◽  
Input Resistance ◽  
Field Potentials ◽  
Maximal Amplitude

1. Extracellular and intracellular recordings and measurements of the extracellular concentration of free K+ ([K+]o) were performed in the CA1 subfield of the rat hippocampal slice during perfusion with artificial cerebrospinal fluid (ACSF) in which NaCl had been replaced with equimolar Na-isethionate or Na-methylsulfate (hereafter called low Cl- ACSF). 2. CAl pyramidal cells perfused with low Cl- ACSF generated intracellular epileptiform potentials in response to orthodromic, single-shock stimuli delivered in stratum (S.) radiatum. Low-intensity stimuli evoked a short-lasting epileptiform burst (SB) of action potentials that lasted 40–150 ms and was followed by a prolonged hyperpolarization. When the stimulus strength was increased, a long-lasting epileptiform burst (LB) appeared; it had a duration of 4–15 s and consisted of an early discharge of action potentials similar to the SB, followed by a prolonged, large-amplitude depolarizing plateau. The refractory period of the LB was longer than 20 s. SB and LB were also seen after stimulation of the alveus. 3. Variations of the membrane potential with injection of steady. DC current modified the shape of SB and LB. When microelectrodes filled with the lidocaine derivative QX-314 were used, the amplitudes of both SB and LB increased in a linear fashion during changes of the baseline membrane potential in the hyperpolarizing direction. The membrane input resistance, as measured by injecting brief square pulses of hyperpolarizing current, decreased by 65-80% during the long-lasting depolarizing plateau of LB. 4. A synchronous field potential and a transient increase in [K+]o accompanied the epileptiform responses. The extracellular counterpart of the SB was a burst of three to six population spikes and a small increase in [K+]o (less than or equal to 2 mM from a resting value of approximately 2.5 mM). The LB was associated with a large-amplitude, biphasic, negative field potential and a large increase in [K+]o (up to 12.4 mM above the resting value). Changes in [K+]o during the LB were largest at the border between S. oriens and S. pyramidale. This was also the site where the field potentials measured 2–5 s after the stimulus attained their maximal amplitude. Conversely, field potentials associated with the early component of the LB or with the SB displayed a maximal amplitude in the S. radiatum. 5. Spontaneous SBs and LBs were at times recorded in the CA1 and in the CA3 subfield.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals A Novel Antiinflammatory Maintains Glucocorticoid Efficacy with Reduced Side Effects

2003 ◽  
Vol 17 (5) ◽  
pp. 860-869 ◽  
Author(s):  
Michael J. Coghlan ◽  
Peer B. Jacobson ◽  
Ben Lane ◽  
Masaki Nakane ◽  
Chun Wei Lin ◽  
...  
Keyword(s):  
Side Effects ◽  
Inflammatory Disease ◽  
Target Genes ◽  
Negative Effects ◽  
Interacting Protein ◽  
Altered Gene ◽  
Antiinflammatory Effects

Abstract Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor γ coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

Spike Timing of Lacunosom-Moleculare Targeting Interneurons and CA3 Pyramidal Cells During High-Frequency Network Oscillations In Vitro

2007 ◽  
Vol 98 (1) ◽  
pp. 96-104 ◽  
Author(s):  
Jay Spampanato ◽  
Istvan Mody
Keyword(s):  
High Frequency ◽  
Epileptiform Activity ◽  
Field Potential ◽  
Pyramidal Cells ◽  
Gamma Oscillations ◽  
Spike Timing ◽  
Network Activity ◽  
Network Oscillations ◽  
Ca3 Pyramidal Cells

Network activity in the 200- to 600-Hz range termed high-frequency oscillations (HFOs) has been detected in epileptic tissue from both humans and rodents and may underlie the mechanism of epileptogenesis in experimental rodent models. Slower network oscillations including theta and gamma oscillations as well as ripples are generated by the complex spike timing and interactions between interneurons and pyramidal cells of the hippocampus. We determined the activity of CA3 pyramidal cells, stratum oriens lacunosum-moleculare (O-LM) and s. radiatum lacunosum-moleculare (R-LM) interneurons during HFO in the in vitro low-Mg2+ model of epileptiform activity in GIN mice. In these animals, interneurons can be identified prior to cell-attached recordings by the expression of green-fluorescent protein (GFP). Simultaneous local field potential recordings from s. pyramidale and on-cell recordings of individual interneurons and principal cells revealed three primary firing behaviors of the active cells: 36% of O-LM interneurons and 60% of pyramidal cells fired action potentials at high frequencies during the HFO. R-LM interneurons were biphasic in that they fired at high frequency at the beginning of the HFO but stopped firing before its end. When considering only the highest frequency component of the oscillations most pyramidal cells fired on the rising phase of the oscillation. These data provide evidence for functional distinction during HFOs within otherwise homogeneous groups of O-LM interneurons and pyramidal cells.

scholarly journals Beyond spikes: Multiscale computational analysis of in vivo long-term recordings in the cockroach circadian clock

2019 ◽  
Vol 3 (4) ◽  
pp. 944-968 ◽  
Author(s):  
Pablo Rojas ◽  
Jenny A. Plath ◽  
Julia Gestrich ◽  
Bharath Ananthasubramaniam ◽  
Martin E. Garcia ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Field Potential ◽  
Dynamic Features ◽  
Pigment Dispersing Factor ◽  
Gamma Frequency ◽  
Accessory Medulla ◽  
Electrophysiological Recordings ◽  
Wide Range

The circadian clock of the nocturnal Madeira cockroach is located in the accessory medulla, a small nonretinotopic neuropil in the brain’s visual system. The clock comprises about 240 neurons that control rhythms in physiology and behavior such as sleep-wake cycles. The clock neurons contain an abundant number of partly colocalized neuropeptides, among them pigment-dispersing factor (PDF), the insects’ most important circadian coupling signal that controls sleep-wake rhythms. We performed long-term loose-patch clamp recordings under 12:12-hr light-dark cycles in the cockroach clock in vivo. A wide range of timescales, from milliseconds to seconds, were found in spike and field potential patterns. We developed a framework of wavelet transform–based methods to detect these multiscale electrical events. We analyzed frequencies and patterns of events with interesting dynamic features, such as mixed-mode oscillations reminiscent of sharp-wave ripples. Oscillations in the beta/gamma frequency range (20–40 Hz) were observed to rise at dawn, when PDF is released, peaking just before the onset of locomotor activity of the nocturnal cockroach. We expect that in vivo electrophysiological recordings combined with neuropeptide/antagonist applications and behavioral analysis will determine whether specific patterns of electrical activity recorded in the network of the cockroach circadian clock are causally related to neuropeptide-dependent control of behavior.

scholarly journals Dissociated Steroids

2007 ◽  
Vol 7 ◽  
pp. 421-430 ◽  
Author(s):  
Matthew C. Catley
Keyword(s):  
Side Effects ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Therapeutic Index ◽  
Clinical Use ◽  
Response Elements ◽  
Activation Of Transcription

Glucocorticoids (GCs) are some of the most important drugs in clinical use today. They are mainly used to suppress disease-related inflammation and are widely used for the treatment of many inflammatory diseases including asthma and arthritis. However, GCs are also associated with debilitating side effects that place limitations on the long-term use of these drugs. The development of a GC with reduced side effects would allow more effective treatments for patients who require long-term suppression of inflammation. GCs exert their effects by binding and activating the GC receptor (GR). The activated receptor then binds GC response elements (GREs) in the promoter of genes, and activates transcription (transactivation) or interferes with the activation of transcription by inhibiting the transactivating function of other transcription factors, such as AP-1 and NF-ĸB (transrepression). Transrepression is believed to be responsible for the majority of the beneficial anti-inflammatory effects of GCs, whereas transactivation is believed to play a bigger role in the unwanted side effects of GCs. Compounds that can dissociate the transactivation function of GCs from the transrepression function may, therefore, have an improved therapeutic index. A number of these dissociated corticosteroids have been developed.In vitroassays using these compounds appear to show good dissociation. However,in vivo, the dissociation appears to be lost and these compounds still produce many of the side effects associated with conventional GCs. A better understanding of the molecular mechanisms behind GC-induced effects would allow the design of novel selective GR modulators with an improved therapeutic index.

Sign in / Sign up

Export Citation Format

Share Document