Coordinator Steph Wetherell explains why that brought them to us Despite a night of atrocious weather, forty representatives of Greater Manchester food organisations made it to Bridge 5 Mill at the end of October for an evening of wine, catching up and a gorgeous photo farmer exhibition to launch the Sustainable Food Vision for Greater Manchester. Former members of the Kindling team, Alex and Bailey were instrumental in co-ordinating the FarmStart programme and developing and running Veg Box People.
In March they left for pastures new literally , to a five acre organic farm in Devon. Current Kindling member Lyndsey, who is participating in our Commercial Growers Course, visited them there at Haye Farm during October to lend a hand on the land, have a good catch up, and learn about their growing journey so far Where has the time gone? Incredibly fast, and it feels like I have only just touched the surface on learning about Stockfree Organic Growing.
My 1st year is nearly coming to an end and it has been amazing and hard work. Learning to be observant, keeping detailed records, being creative on how you see things. Not to get stuck into one way of doing and also to communicate and work within the team. The Kindling Trust is a not for profit social enterprise with charitable aims Company number: Search form Search. Multilingual Communication's Student Rahel from Cologne spent four months with us. Making the most of her time in Manchester, from pruning apple tree's to scaling mountains in the peak district.
Include in timeline:. A Sample EEG trace illustrating an optogenetically evoked electrographic seizure, with automatically detected seizure start and end indicated by vertical dashed lines see Methods and Supplementary Fig. Note that seizure begins with rapid oscillations and ends with slow oscillations. Laser-light stimulation bouts are indicated in blue Fig. B Wigner transform of electrographic seizure in A showing graded decay in high-frequency components as well as a gradual increase in low-frequency power.
D Peak power of high frequencies occurred earlier compared with that of low frequencies 6. Data points indicate the difference between the low and high frequency peak times for individual seizures. Box plot shows first quartile, median, and third quartile with whiskers denoting one standard deviation from the mean. Prior studies have revealed that EEG power is often temporarily reduced following electrographic seizures, a notion known as postictal depression In keeping with previous models of epilepsy 26 , we found a period of reduced EEG power following seizures.
We next investigated how different frequency bands were individually altered by the kindling protocol over tens of minutes to days Supplementary Fig. We found that, after the induction Fig. This effect did not, however, persist across stimulation sessions — no changes in the power of any frequency band was observed when comparing the baseline period before induction across sessions Supplementary Fig. This result was in contrast to that found with laser-light evoked EEG responses Fig.
Taken together Fig. Although postictal depression of intrinsic EEG power followed immediately after optogenetically induced seizures Supplementary Fig. S6 , as previously shown 25 , 26 , this outcome depended on which frequency bands and on which time periods were analysed Supplementary Fig. Only the second of paired laser-evoked EEG responses were potentiated in the long term, persisting across different sessions Fig. Based on the classical kindling model 1 , we developed a robust neocortical optogenetic kindling method of epilepsy.
Optokindling recapitulated several essential hallmark features of its classical counterpart 4. First, a majority of animals developed seizures, in line with electrical kindling studies showing robust seizure development 27 , Second, seizures emerged gradually over several stimulation sessions. Similar to electrical kindling, optokindling required more than ten sessions before first seizure and around twenty sessions for the development of generalized seizures 28 , Although some classical reports show generalized seizures earlier than session ten, there is considerable variability from animal to animal This is intriguing, given that electrical kindling stimulates a more diverse population of cells than our optokindling protocol.
Third, once seizures began occurring, they increased in severity and duration across sessions. In addition, the threshold laser stimulation time necessary for evoking seizures decreased 4 , although we did not determine whether this also resulted in an extrafocal threshold reduction Finally, there was a long-term retention of seizure susceptibility in kindled animals.
These findings are consistent with previous reports on the hallmark features of electrical kindling 27 , 32 , As a proof of principle, we used our optogenetic model to demonstrate several findings previously found in epilepsy models. First, we found evidence that high-frequency oscillations precede low-frequency activity in seizures Second, we observed that postictal depression of EEG power was associated with seizures, as previously shown in neocortex Finally, we found that epileptogenesis was associated with a graded change in evoked EEG dynamics, as previously found in local field potentials of kindled hippocampus Optokindling has several advantages compared to its classical electric counterpart.
First, seizures developed in the absence of gross brain damage, providing an experimental epileptogenesis paradigm with improved specificity for plasticity and for pathological activity. In the current study, viral injection was chosen as the method to restrict the population of cells expressing ChR2. This allowed us to more closely mimic the etiology of focal seizures which involves initial recruitment of a smaller network of neurons with subsequent spreading of activity and worsening of seizures. Although the initial viral injection presumably resulted in some degree of injury, we were not able to detect any many weeks afterwards.
In the future, ChR2-expressing transgenic mouse lines could be employed to eliminate any injury to the intact brain. Moreover, different number of cells could be progressively recruited during optokindling in ChR2-expressing transgenic mouse lines to investigate different epileptogenesis scenarios by dilating or restricting illumination area. Craniotomy can also be avoided completely by activating ChR2 through the skull 34 , Although several classical kindling studies also did not report gross tissue damage 19 , the chronic stimulation electrode must leave some damage whereas the fibers we used for optokindling did not penetrate the brain.
Second, optokindling enables targeted cell-type-specific recruitment. This is an important feature since neuronal plasticity depends on synapse type 36 , so kindling is expected to be cell-type dependent. Indeed, directly optogenetically driven seizures have been shown to depend critically on cell type Indiscriminate activation of several types of local neurons and fibers is in other words one drawback of classical electrical kindling, although optokindling will also drive other cell types indirectly.
In the future, elucidating the cell-type dependence in optokindling will help clarify the circuit mechanisms that underpin epileptogenesis. Third, optokindling provides a means for testing the two-hit model with respect to injury and inflammation. In the two-hit view on epilepsy, a second agent is required for spontaneous seizures to develop However, it is difficult to explore the involvement of inflammation and injury with classical electrical kindling, since the stimulation electrode introduces those two factors.
With optokindling, in the absence of craniotomy or viral infection, inflammation could be systematically added as a second factor to investigate how it promotes epileptogenesis. It is unclear why the expected spontaneous seizures seen in epilepsy are not observed with optokindling. However, it is noteworthy that spontaneous seizures have not been observed with classical electrical kindling either — unless the animal is over-kindled through hundreds of sessions 15 , This may imply that spontaneous seizures develop slowly.
Alternatively, over-kindling with the classical protocol may contribute additional and possibly critically needed factors, e. Optokindling may provide improved experimental control suitable for investigating the fundamental question of what is required to achieve spontaneous seizures. Another limitation is that our optogenetic paradigm is time consuming. To enable us to monitor the gradual emergence of seizures, we spaced stimulation sessions by two days. We were also motivated by a concern that stimulation sessions spaced too closely might lower the efficacy of epileptogenesis 27 , Although this approach provided gradual emergence of evoked seizures, which was desirable for studying epileptogenesis, the long delay between sessions required more than 50 days of repeated spaced stimulation, which is not ideal for many applications.
We note that this caveat is essentially shared with the classical electrical kindling model. Additional work is required to narrow down the ideal parameter space for rapid optokindling. We demonstrated that due to repeated pathological activation of a small cluster of PCs in motor cortex, local circuits undergo plastic changes that lead to the appearance of generalized seizures.
This circuit plasticity appeared to happen in the absence of gross brain damage and inflammation, implicating plasticity as key causative agent. Although all investigated EEG frequency bands were unaffected across sessions, we found long-term alterations of evoked EEG dynamics, additionally supporting the interpretation that plasticity at least partially underpinned the seizure-promoting circuit changes.
However, since the initial amplitude of evoked EEG responses was unaffected, this did not appear to be a form of Hebbian plasticity 2. Although we were inspired by classical LTP protocols in designing our optogenetic protocol, our study did not directly address how Hebbian LTP related to epileptogenesis 4 , 6 , which would have required testing if kindling occluded subsequent LTP induction 13 , We also explored whether optokindling was associated with changes in PC intrinsic properties, but found none, consistent with previous reports 40 , There were, however, marked changes in short-term dynamics of evoked EEG responses, which could have been due to alterations in synaptic short-term plasticity, or to reduced inhibitory feedback Plasticity was thus linked to epileptogenesis in our model, although the specific nature of this plasticity remains to be uncovered.
To our knowledge, our model is the first to systematically use optogenetics for neocortical kindling of awake behaving and otherwise healthy animals. One recent hippocampal study demonstrated the graded development of seizures using optogenetics 42 , but it did not explore if the elevated seizure susceptibility was retained in the long term like in the original kindling model 1 , nor was it possible to evaluate the behavioral component since the mice were sedated.
There have also been several studies using optogenetics to halt 43 , 44 , 45 , 46 and initiate seizures in hippocampus 47 , 48 as well as cortex 26 , Although these studies demonstrated optogenetically elicited seizures, they did not show kindling, i. Also, these studies relied on optogenetic stimulation either in combination with classical induction models 45 , 50 or with pre-existing disease phenotypes 49 , thus making it difficult to disentangle the role of plasticity from that of injury and inflammation in seizure development.
However, with optokindling, it is possible to isolate the distinct role of plasticity in epileptogenesis. To sum up, even though kindling does not model all variants of epilepsy equally well 5 , it is with optokindling possible to circumvent several limitations associated with other seizure induction models, e. Future studies with optokindling may explore cell type specific contributions to epileptogenesis, or microcircuit plasticity associated with epileptogenesis, in addition to the roles of injury and inflammation in the two-hit model. Because of its more specific focus on plasticity, we believe optokindling will be useful for finding therapeutic treatments, to halt or slow down pathological plasticity in epileptogenesis.
For collection of acute slices, mice were anesthetized with Avertin Sigma Aldrich, Oakville, ON, Canada or isoflurane and sacrificed once the hind-limb withdrawal reflex was lost. Every attempt was made to ensure minimum discomfort of the animals. Males were chosen solely instead of females since the estrus cycle can affect seizure susceptibility Injection coordinates relative to bregma were 1.
An Optogenetic Kindling Model of Neocortical Epilepsy
We subsequently placed and cemented 1. Animals were evaluated for correct placement with enhanced yellow fluorescent protein EYFP staining see below. We used a separate ground reference screw for the left and right hemisphere.
Animals were given 21 days to recover from surgery after which, they were habituated for three days to the recording setup before commencing stimulation sessions. To reduce noise by serving as a Faraday cage, the copper plate and mesh of the cage were connected to the amplifier chassis ground. The implanted ferrule did not penetrate the brain. This computer also episodically TTL-gated the two nm lasers. Animal behavior was recorded with two cameras, one above the recording setup, and one to the side Logitech C webcam, Tiger Direct.
Video was acquired using iSpy software Version 6. If behavior straddled two Racine stages, the score was taken as the intermediate value, e. As a form of verification, the Racine score was determined independently of the automated electrographic seizure detection. We did not categorize paw movement due to direct optogenetic activation as clonus, since it is a trivial and direct consequence of activation of motor programs that is unrelated to epilepsy.
Animals without seizures were confirmed to have viral expression by staining for EYFP and correct placement was verified with EEG responses due to laser light stimulation. Stimulation sessions were numbered sequentially starting at one. Each session consisted of an initial minute-long baseline period, a three-minute-long induction, and a second minute-long baseline. We categorized frequency bands as follows e. Supplementary Fig. We employed two types of control animals. In ChR2 controls, the same ChR2 construct as stimulated animals was expressed, but the Hz stimulation was omitted from the induction period; the baseline stimulation pattern was as above.
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In rekindling experiments, mice were not stimulated for 36 days after the last of the initial 25 kindling sessions. Once stimulation was resumed, mice were stimulated every two days, as before. The first session after the day-long pause was considered rekindling session one, with subsequent sessions numbered sequentially. Goddard, G. Development of epileptic seizures through brain stimulation at low intensity. Nature , — Hebb, D. The Organization of Behavior. Wiley, Markram, H. A history of spike-timing-dependent plasticity.
Teskey, G. In Toward a the or y of neuroplasticity eds Shaw, C. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research.
Morimoto, K. Kindling and status epilepticus models of epilepsy: rewiring the brain. Cavazos, J. Neuronal loss induced in limbic pathways by kindling: evidence for induction of hippocampal sclerosis by repeated brief seizures. The Journal of Neuroscience 14 , — Berndt, A. High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Cardin, J. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Aravanis, A. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology.
Boyden, E. Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8 , — Cain, D. Kindling in sensory systems: neocortex. Experimental neurolog y 7 6, —, 82 [pii] Rate, timing, and cooperativity jointly determine cortical synaptic plasticity. Neuron 32 , — Abrahamsson, T. In-vitro investigation of synaptic plasticity.
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Pinel, J. Experimental epileptogenesis: kindling-induced epilepsy in rats.
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Experimental neurology 58 , — Michael, M. Development of spontaneous seizures over extended electrical kindling: I. Electrographic, behavioral, and transfer kindling correlates. Racine, R. Modification of seizure activity by electrical stimulation. Motor seizure. Electroencephalogr Clin Neurophysiol 32 , — Quigg, M.
Epilepsia 41 , — From traumatic brain injury to posttraumatic epilepsy: what animal models tell us about the process and treatment options. Turrigiano, G. The dialectic of Hebb and homeostasis. Wierenga, C. Miniature inhibitory postsynaptic currents in CA1 pyramidal neurons after kindling epileptogenesis. J Neurophysiol 82 , — Reyes, A. Journal of Neuroscience 19 , — Cudmore, R. Long-term potentiation of intrinsic excitability in LV visual cortical neurons. J Neurophysiol 92 , — Worrell, G. High-frequency oscillations and seizure generation in neocortical epilepsy.
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Ayala, G. Excitability changes and inhibitory mechanisms in neocortical neurons during seizures. Khoshkhoo, S. A permanent change in brain function resulting from daily electrical stimulation. Wada, Y. Jpn J Psychiat Neur 43 , — Lothman, E. Modification of seizure activity by electrical stimulation: cortical areas. Electroencephalogr Clin Neurophysiol 38 , 1—12 Scott, B. Extrafocal threshold reductions in amygdala-kindled rats. Wada, J. Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats. Epilepsia 15 , — Dennison, Z. Persistence of kindling: effect of partial kindling, retention interval, kindling site, and stimulation parameters.
Epilepsy Res 21 , — Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin Lin, J. ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Larsen, R.