Posted comment on ´Frequency-selective control of cortical and subcortical networks by central thalamus` by J. Liu, H.J. Lee, A.J. Weitz, Z. Fang, P. Lin, M. Choy, R. Fisher, V. Pinskiy, A. Tolpygo, P. Mitra, N. Schiff and J.H. Lee published in eLife 2015;4:e09215 (doi.org/10.7554/eLife.09215)
The authors of this paper explored the network connections of the central thalamus which is known to play a role in arousal and organized behaviour. They used optogenetics (20 s periods of light stimulation every minute for 6 min at 10, 40, or 100 Hz) with fMRI to form the ofMRI technique which provided whole brain spatial and temporal information. A stereotactic injection was given to the subject in the right CL and PC intralaminar nuclei of central thalamus with the adeno-associated virus carrying channelrhodopsin-2 (ChR2) and the fluorescent reporter protein EYFP under the control of the CaMKIIa promoter. This promoter was used since it is expressed primarily in excitatory neurons which in the thalamus are mostly relay cells. Liu and colleagues found that nearly 34% of cells were EYFP-positive, co-expressing CaMKIIa which showed that the technique was highly selective for excitatory neurons and hence ideal for neuronal stimulation experiments. Targeted stimulation of the intralaminar nuclei area was achieved by MR-validated stereotactic fiber placement and using a small volume of excited tissue. Electrophysiology and video EEG monitoring was also used to investigate the network connections. Ex vivo fluorescence microscopy images of ChR2-EYFP expression were also carried out.
Liu and colleagues found in their experiments that EYFP-expressing axons could be seen throughout the forebrain, including areas such as the frontal cortex and striatum with the medial prefrontal, lateral prefrontal, cingulate, motor, and sensory cortices all receiving strong projections from the thalamus. Input was found to be highly convergent at the superficial layers, with moderate but weaker projections also present in the middle layers. Furthermore, projections were significantly restricted to the hemisphere ipsilateral to the virus injection for both the cortex and striatum.
The authors also found using the ofMRI technique at all 3 frequencies strong positive blood-oxygen-level-dependent (BOLD) signals at the site of stimulation that was highly synchronized to light delivery, increased upon optical activation, and gradually returned to baseline following the end of stimulation. Local neuronal firing was also observed. A much larger volume of brain tissue was activated by stimulation at 40Hz and 100 Hz compared to 10 Hz as was the frontocortical areas and striatum in particular. The difference in activation volume between the low 10 Hz stimulation and the higher 40 or 100 Hz stimulation frequencies was significant for the thalamus, striatum, and medial prefrontal, lateral prefrontal, cingulate, motor, and sensory cortical areas. Striatal activity was found to be primarily localized to the dorsal sector, with negligible activity occurring in the ventral region and BOLD activation was generally restricted to the ipsilateral hemisphere, although activation volumes in the contralateral striatum, lateral prefrontal cortex, motor cortex, and sensory cortex were all significantly greater during 100 Hz stimulation compared to the low 10 Hz stimulation. The rapid 40 and 100HZ stimulations of the central thalamus causing the widespread activation of the forebrain caused a state of arousal in the sleeping rats and the increase in neuronal firing rate observed during the 100 Hz stimulation was generally maintained throughout the 20 s stimulation period.
With the slower 10Hz stimulation, Liu and colleagues found that even though the excitatory neurons had been targeted for activation the somatosensory cortex exhibited a strong negative BOLD signal during 10 Hz stimulation which suggested that baseline activity had been suppressed. This was supported by the results of the ofMRI technique which showed that 10 Hz stimulation had decreased the neuronal firing rate between pre-stimulation and stimulation period and this decrease occurred mainly between 5 to 15 s after initiation of the stimulation. Spiking events which occurred during this inhibition had a non-uniform distribution over time suggesting that only sometimes did the glutaminergic thalamocortical input generate action potentials. The resulting lower activation of the forebrain and inhibition of the sensory cortex led to seizure-like unconsciousness of the test subject.
Using the ofMRI technique, Liu and colleagues could identify a group of inhibitory neurons in the central thalamus in the zona incerta (ZI) region which sends direct GABAergic projections to the somatosensory thalamic nuclei and sensory cortex and whose activity is linked to whisker stimulation. The authors found that the majority of the ZI cells exhibited increases in firing rate during the central thalamus stimulation at 10Hz and 40Hz. Spindle like oscillations (SLOs) were evoked at the lower 10Hz stimulation, but not at 40Hz and these oscillations exhibited an inter-event interval centered around 6.6 s similar to those observed in the thalamus during the onset of sleep. The suppressed ZI firing during the 10Hz stimulation was found to lead to a reduction of evoked cortical inhibition. Simultaneous EEG recordings in the frontal cortex revealed strong spike-wave modulation during the 10 Hz stimulation associated with the loss of consciousness and lower amplitude, fast oscillations during 40 Hz stimulation associated with aroused brain states.
Liu and colleagues investigated if the evoked activity in ZI plays a causal role in driving the frequency-dependent inhibition of the somatosensory cortex. They injected the inhibitory opsin halorhodopsin (eNpHR) fused to the mCherry fluorescent marker and controlled by the pan-neuronal hSyn promoter into the ZI of four animals expressing ChR2-EYFP in the central thalamus. The light stimulation at 10Hz of halorhodopsin was found to be successful in suppressing ZI activity and this had a net inhibitory effect on somatosensory cortex activity. The authors suggested that this was brought about by hyperpolarization of the neuronal cells in this area.
The results found with ofMRI were supported by the simultaneous video and EEG recordings. During the 10 Hz stimulation, the majority of animals exhibited behavior indicative of an absence seizure, including freezing and behavioral arrest throughout stimulation leading to sleep onset. The most common EEG response was a shift to slow spike-wave discharges indicative of a loss of consciousness. The higher 40 and 100 Hz stimulations led to the awake state and an EEG pattern associated with cortical activation and desynchronization.
Therefore, the authors concluded that the awake or unconscious (or sleep) state is promoted by the ZI area of the central thalamus and how fast these neurons are stimulated. Differences in time could reflect the short-term plasticity of the thalamocortical pathway which has frequency-dependent properties. Their experiments show that neuronal cells in a single population can have different firing patterns and promote different effects on connecting areas depending on the temporal code of their stimulation. Since there are GABAergic projections from the ZI to central thalamus, activity in ZI may also limit forebrain activation through incertal-thalamic feedback. Therefore, the hypothesized feedforward and feedback inhibition via ZI both suggest a direct projection from central thalamus to ZI, which the fluorescence imaging data supported. However, there is no thalamic input specifically from the intralaminar nuclei to ZI and therefore arousal regulation is driven by the central thalamus which has a causal and frequency-dependent influence on ZI. Suppression of the ZI activity modulates the activity of the overall brain which is susceptible to thalamus stimulation eg. inhibitory signals from the ZI lead to frequency-dependent depression of cortical activity. This type of information can be important in the treatment of traumatic brain injury and the minimization of cognitive defects.
What makes this paper interesting is the use of the newly popular technique of optogenetics to further investigate a brain area with relation to a well-known function. It has been known for a long time that the central thalamus is an important area relating to arousal/alertness and sleep/wakefulness and that damage to this area can be lead to not only excessive sleeping and coma, but also cognitive problems such as loss of memory. The study described here in this Blog post uses optogenetics to investigate the arousal and sleep function of the thalamus further. It can be seen that the central thalamus and intraluminar nuclei when stimulated at low frequencies leads to the subject losing consciousness, limited forebrain functioning, strong inhibition of the somatosensory cortex and EEG spindle bursts. Alternatively, high frequency stimulation leads to arousal of the subject, attention and goal directed behaviour and is supported by desynchronized EEG cortical signals.
Using optogenetics with its high sensitivity to spatial and temporal changes, these different effects can be attributed to activity in a specific thalamus region, that of the zona incerta (ZI). This is a grey matter area located in the subthalamus under the thalamus and gates sensory input and synchronized cortical and subcortical brain rhythms. It is known that this area has a wide variety of cells all merging areas into one another and is divided into sectors eg. rostral, dorsal, ventral (known to be GABergic cells) and caudal known as the ´motor sector` and an area bringing research attention because of targeting by tDCS in sufferers of Parkinson`s disease.
ZI is also known to have numerous connections some outgoing (eg. to cerebral cortex, hypothalamus), others incoming (eg. cingulate cortex, frontal lobe, parietal lobe, cerebellum, raphe nuclei, thalamic reticular nucleus, super colliculus, the last three being cholinergic) and some bidirectional such as the thalamus (eg. intraluminar and central lateral nucleus) substantia nigra (linked to DOPA and Parkinson`s disease) and globus pallidus (linked to reward). The capability of the area appears to be linked to the frequency at which it and the thalamus are stimulated. The stimulation either removes the inhibition placed upon the area (high frequency) or activates it (low frequency). Sensory suppression means hyperpolarization of thalamus leading to GABAergic IPSP and depression in the ZI area. Sensory activation means likely glutaminergic depolarization of the thalamus leading to EPSP of the ZI. Hence, depression of ZI is inhibited by the depolarization of the thalamus. Therefore, the optogenetics study of Liu and colleagues shows that the frequency of stimulation has a wide-ranging neuronal firing affect. Similar to work on the medial leminiscus tract and the thalamus, frequency of stimulation changes subsequent firing such as short EPSP leads to longer IPSP (Castro-Alamancos). Further investigation of the firing within smaller frequency ranges is likely to reiterate the results of Bartho et al. who used anaesthetized rats. They showed that slow cortical 1-3HZ waves become synchronized to depth-negative phasing of cortical waves to a degree comparable to thalamocortical neurons; paroxysomal high voltage spindles display highly rhythmic activity in tight synchrony with cortical oscillations; and 5-9HZ oscillations respond with a change in interspike interval distribution. Hence, the optogenetics technique can be used to further investigate the neural networks existing in the brain and the effect on firing of specific frequency stimulation.
However, herein lies some problems with optogenetics. Is this technique only repeating, albeit more accurately, studies that were carried out many years in the past? We may be able to pinpoint areas more accurately and say where and with what these areas are networking, but does that add to previous knowledge to sufficiently answer the questions about how memory and consciousness are formed for example? Or how neurodegenerative diseases start? Optogenetics is expensive, there are small sample numbers and the technique has an element of risk with human subjects. Plus it requires cell alterations (the neurons have to express the gene encoding the light sensitive ion channel) so can we guarantee that what we are seeing is actually real and not the result of this insertion? The benefit of this technique could be in cases where it is linked with other techniques such as cell targeting of chemotherapy drugs or in cases like Parkinson`s disease where we can override the effects of limited DOPA in one area and consequential reduced firing by stimulating with light the next area in the motor system. Another benefit of the technique could be in cases where we can compare the molecular complexity of mechanisms investigated by other means for areas lit up due to firing from the targeted area. It is clear that the technique is here to stay and can offer new experimental avenues to explore, but the talked about panacea for human mental disorders is in my opinion not yet proven.
Since we`re talking about the topic……………………….
………………..if Alzheimer`s disease is linked to hyperexcitability of the hippocampus, could optogenetics with illumination at intervals be used to suppress activation in this area and hence, reduce the build-up of beta amyloid?
………………could the use of gold nanoparticles attached to specific antibodies as suggested by Bezanilla instead of gene therapy be used to study other membrane molecules where the transport of electrons is a part of their function and not just neurons?