Current Biology
Magazine challenge to our understanding of this process. Development of a robust in vitro spermatogenesis system for mammals would provide useful insights. So far, numerous approaches using the entire tissue or separated single cells from the testis in 2D or 3D cell culture systems have been explored, which is encouraging. Additionally, a robust in vitro spermatogenesis system might help define the causes of infertility in humans. FURTHER READING Ashrafzadeh, A., Karsani, S.A., and Nathan, S. (2013). Mammalian sperm fertility related proteins. Int. J. Med. Sci. 10, 1649–1657. Ellis, R.E., Stanfield, G.M. (2014). The regulation of spermatogenesis and sperm function in nematodes. Semin. Cell Dev. Biol. 29, 17–30. Foster, J.A., and Gerton, G.L. (2016). The acrosomal matrix. Adv. Anat. Embryol. Cell Biol. 220, 15–33. Gleason, E.J., Hartley, P.D., Henderson, M., HillHarfe, K.L., Price, P.W., Weimer, R.M., Kroft, T.L., Zhu, G.D., Cordovado, S., and L’Hernault, S.W. (2012). Developmental genetics of secretory vesicle acidification during Caenorhabditis elegans spermatogenesis. Genetics 191, 477–491. Griswold, M.D. (2016). Spermatogenesis: the commitment to meiosis. Physiol. Rev. 96, 1–17. Hunter, D., Anand-Ivell, R., Danner, S., and Ivell, R. (2012). Models of in vitro spermatogenesis. Spermatogenesis 2, 32–43. Jan, S.Z., Hamer, G., Repping, S., de Rooij, D.G., van Pelt, A.M., and Vormer, T.L. (2012). Molecular control of rodent spermatogenesis. Biochim. Biophys. Acta 1822, 1838–1850. Miao, L., and L’Hernault, S.W. (2014). Role of posttranslational modifications in C. elegans and Ascaris spermatogenesis and sperm function. Adv. Exp. Med. Biol. 759, 215–239. Morgana, D.E., Crittendenb, S.L., and Kimble, J. (2010). The C. elegans adult male germline: stem cells and sexual dimorphism. Dev. Biol. 346, 204–214. Nishimura, H., and L’Hernault, S.W. (2010). Spermatogenesis-defective (spe) mutants of the nematode Caenorhabditis elegans provide clues to solve the puzzle of male germline functions during reproduction. Dev. Dyn. 239, 1502–1514. Nishimura, H., and L’Hernault, S.W. (2016). Gamete interactions require transmembranous immunoglobulin-like proteins with conserved roles during evolution. Worm 5, e1197485. O’Donnell, L. (2014). Mechanisms of spermiogenesis and spermiation and how they are disturbed. Spermatogenesis 4, e979623. Rato, L., Alves, M.G., Socorro, S., Duarte, A.I., Cavaco, J.E., and Oliveira, P.F. (2012). Metabolic regulation is important for spermatogenesis. Nat. Rev. Urol. 9, 330–338. Sato, T., Katagiri, K., Kojima, K., Komeya, M., Yao, M., and Ogawa, T. (2015). In vitro spermatogenesis in explanted adult mouse testis tissues. PLoS One 10, e0130171. Vacquier, V. (1998). Evolution of gamete recognition proteins. Science 281, 1995–1998. Yan, W. (2009). Male infertility caused by spermiogenic defects: lessons from gene knockouts. Mol. Cell Endocrinol. 306, 24–32.
1
Department of Life Science, Setsunan University, Neyagawa, Osaka 572-8508, Japan. 2Department of Biology, Emory University, Atlanta, GA 30322, USA. *E-mail:
[email protected]
R994
Correspondence
Restoring consciousness with vagus nerve stimulation Martina Corazzol1,2,4, Guillaume Lio1,2,4, Arthur Lefevre1,2, Gianluca Deiana1,2, Laurence Tell3, Nathalie André-Obadia3, Pierre Bourdillon3, Marc Guenot2,3, Michel Desmurget1,2, Jacques Luauté2,3,4, and Angela Sirigu1,2,4,* Patients lying in a vegetative state present severe impairments of consciousness [1] caused by lesions in the cortex, the brainstem, the thalamus and the white matter [2]. There is agreement that this condition may involve disconnections in long-range cortico–cortical and thalamo-cortical pathways [3]. Hence, in the vegetative state cortical activity is ‘deafferented’ from subcortical modulation and/or principally disrupted between frontoparietal regions. Some patients in a vegetative state recover while others persistently remain in such a state. The neural signature of spontaneous recovery is linked to increased thalamo-cortical activity and improved fronto-parietal functional connectivity [3]. The likelihood of consciousness recovery depends on the extent of brain damage and patients’ etiology, but after one year of unresponsive behavior, chances become low [1]. There is thus a need to explore novel ways of repairing lost consciousness. Here we report beneficial effects of vagus nerve stimulation on consciousness level of a single patient in a vegetative state, including improved behavioral responsiveness and enhanced brain connectivity patterns. Consistent with an important role of the thalamic-cortical axis for awareness, one study has demonstrated an increased behavioral responsiveness after deep thalamic stimulation limited to the stimulation period [4]. Here we propose to activate the thalamocortical network based on vagus nerve stimulation. The vagus nerve carries somatic and visceral efferents
Current Biology 27, R979–R1001, September 25, 2017 © 2017 Elsevier Ltd.
and afferents distributed throughout the central nervous system, either monosynaptically or via the nucleus of the solitary tract (NTS) [5]. The vagus directly modulates activity in the brainstem and via the NTS it reaches the dorsal raphe nuclei, the thalamus, the amygdala, and the hippocampus [5]. In humans, vagus nerve stimulation increases metabolism in the forebrain, thalamus and reticular formation [6]. It also enhances neuronal firing in the locus coeruleus which leads to massive release of norepinephrine in the thalamus and hippocampus, a noradrenergic pathway important for arousal, alertness and the fight-or-flight response [7]. Following the hypothesis that vagus nerve stimulation functionally reorganizes the thalamo-cortical network, we tested its effects on the cortical activity of a patient lying in a vegetative state for 15 years following traumatic brain injury. Behavioral, electroencephalographic (EEG) and 18F-FDG PET recordings were performed before and after surgical implantation of a vagus nerve stimulator. Stimulation was gradually increased to a maximum intensity of 1.5 mA, and its effects were monitored over six months post-implantation. After one month of stimulation, when intensity reached 1 mA, clinical examination revealed reproducible and consistent improvements in general arousal, sustained attention, body motility and visual pursuit. Scores on the Coma Recovery Scale-Revised (CRS-R) test improved, mostly in the visual domain, as stimulation increased, from a score of 5 at baseline (last exam) to 10 at highest intensities (1.00–1.25 mA), indicating a transition from a vegetative to minimally conscious state. Scalp EEG data comparing the patient’s resting state pre- and poststimulation revealed a significant increase in theta band (4–7 Hz) power (one-sample t-test, p < 0.0001 false discovery rate corrected, Figure S1A,B in Supplemental Information, published with this article online), a brain signal found to reliably distinguish minimally conscious patients from vegetative ones [8]. Data driven blind source separation analysis identified eight sources responsible for the stimulation-induced power increase, distributed over the occipito-parietal, inferior temporal and fronto-central regions in addition to a
Current Biology
Magazine A
0.07
Pre-VNS
Pre-VNS
B
0.039 0.036
Median wSMI
wSMI
0.06 0.05 0.04 0.03
0.1
0.08
Mean wSMI
0.07
**
0.05 0.04 0.03 0.02 0.01 0
0.025 0.023
*
Pre-VNS Post-VNS
0.08
10 9
0.06
0.028
0.018
0.09
11
0.031
0.02
0.1
Pre-VNS Post-VNS
12
Median wSMI
0.09
CRS-R
C
0.01
Post-VNS
Post-VNS
0.02
0.033
8 7 6 5
0.07 0.06 0.05 0.04 0.03 0.02
4
0.01
3
0.025 0.03 0.035 0.04 0.045
0.05 0.055 0.06
Mean wSMI
0
Figure 1. Information sharing increases after vagus nerve stimulation over centroposterior regions. (A) Sagittal (left) and coronal (right) views of weighted symbolic mutual information (wSMI) shared by all channels pre- and post-vagus nerve stimulation (VNS) (top and bottom, respectively). For visual clarity, only links with wSMI higher than 0.025 are shown. (B) Topographies of the median wSMI that each EEG channel shares with all the other channels pre- and post-VNS (top and bottom, respectively). The bar graph represents the median wSMI over right centroposterior electrodes (darker dots) which significantly increases post-VNS (permutation test over sessions: Wilcoxon test, p = 0.0266). (C) Localization of the most VNS-reactive theta source showing significant increase of information sharing post-VNS. Sources’ localization is presented over the patient’s cortical surface (probability map, sLoreta current source density: light blue scale) combined with his FDG-PET metabolism (gray scale) as measured three months post-VNS. The source was localized in the inferior parietal lobule. The bar graph represents the mean wSMI shared with all other selected sources pre- and post-VNS (dark gray and light gray, respectively) (permutation test over sessions: Wilcoxon test, p < 0.01 Bonferroni corrected). CRS-R clinical score increased as a function of information sharing over a cortical posterior theta network (Robust regression, p = 0.0015).
deeper source most likely localized in the insula (Figure S1C). These areas belong to the default mode network whose activity appears to reflect the degree of consciousness in non-communicative brain-damaged patients [1]. The highest stimulation intensity induced an increase in theta power over the right inferior parietal and parieto-temporal-occipital border (Figure S1C), a region labelled as a hot-zone for conscious awareness [9]. We calculated weighted symbolic mutual information (wSMI), a measure of cortical connectivity and information sharing known as a sensitive index of consciousness. This measure captures activity in different frequency bands and robustly discriminates the vegetative from minimally conscious state within theta frequency signals (4–10 Hz) [10].
By using a parameter of = 32 ms we found that the median wSMI across channel pairs was significantly higher after vagus nerve stimulation over a parietal region within a cluster including the right centro-temporo-occipital electrodes (Figure 1A,B). The wSMI procedure was also applied on sources previously identified by blind source separation as reactive to vagus nerve stimulation. The purpose here was to isolate regions that most contributed to information sharing, thus suppressing sources with interfering noise activity. Cortical areas showing significant increase in mutual information after stimulation included the inferior parietal, precuneus, posterior cingulate and pre-motor/motor regions (Figure 1C
and Figure S1E). The highest mean wSMI value was found in the inferior parietal cortex/intraparietal sulcus (Figure 1C). This global increase in mean wSMI over theta sources was significantly correlated to the CRS-R scores of clinical improvement (Robust regression, t (-0.0128;3.9436), dfe = 14, p = 0.0015, Figure 1C). These results demonstrate that vagus nerve stimulation enhances information sharing within a centro-posterior network. This is consistent with the observation reported on a large population of brain injured patients showing that activity within centroparietal regions constitute the marker of a conscious state [10]. Importantly, they demonstrate the critical role of the parietal cortex as a central hub for broadcasting neural signals among posterior and central sites in order to strengthen consciousness. Finally, 18F-FDG PET results corroborated EEG findings by showing extensive increases of activity in occipito-parieto-frontal and basal ganglia regions as early as three months after implantation of the stimulator. Vagus nerve stimulation enhanced the metabolic signal in the thalamus, a target of the vagus nerve (Figure S1D). These findings show that stimulation of the vagus nerve promoted the spread of cortical signals and caused an increase of metabolic activity leading to behavioral improvement as measured with the CRS-R scale and as reported by clinicians and family members. Thus, potentiating vagus nerve inputs to the brain helps to restore consciousness even after many years of being in a vegetative state, thus challenging the belief that disorders of consciousness persisting after 12 months are irreversible [1]. The direct connection between the NTS where the vagus nerve originates and the thalamus may be at the origin of the significant increase in theta signal recorded at the cortical level. In particular, the parietal cortex appears to be a major player in guiding the expansion of neural activity across brain areas. The enhanced neural activity might also be mediated by neurotransmission changes given that vagus nerve projections target key regions important for the liberation of norepinephrine and serotonin [7].
Current Biology 27, R979–R1001, September 25, 2017 R995
Current Biology
Magazine Finally, since the vagus nerve has bidirectional control over the brain and the body, reactivation of sensory/ visceral afferences might have enhanced brain activity within a body/ brain closed loop process. Our study demonstrates the therapeutic potential of vagus nerve stimulation to modulate large-scale human brain activity and alleviate disorders of consciousness. SUPPLEMENTAL INFORMATION Supplemental Information contains one figure, one table, experimental procedures, additional discussion, and can be found with this article online at http://dx.doi.org/10.1016/j. cub.2017.07.060. REFERENCES 1. Giacino, J.T., Fins, J.J., Laureys, S., and Schiff, N.D. (2014). Disorders of consciousness after acquired brain injury: the state of the science. Nat. Rev. Neurol. 10, 99-114. 2. Adams, J.H., Graham, D.I., and Jennett, B. (2000). The neuropathology of the vegetative state after an acute brain insult. Brain 123, 1327–1338. 3. Laureys, S. (2005). The neural correlate of (un) awareness: lessons from the vegetative state. Trends Cogn. Sci. 9, 556–559. 4. Schiff, N.D., Giacino, J.T., Kalmar, K., Victor, J.D., Baker, K., Gerber, M., Fritz, B., Eisenberg, B., O’Connor, J., Kobylarz, E.J., et al. (2007). Behavioural improvements with thalamic stimulation after severe traumatic brain injury. Nature 448, 600–603. 5. Rutecki, P. (1990). Anatomical, physiological, and theoretical basis for the antiepileptic effect of vagus nerve stimulation. Epilepsia 31 Suppl 2, S1–S6. 6. Henry, T.R., Votaw, J.R., Pennell, P.B., Epstein, C.M., Bakay, R.A., Faber, T.L., Grafton, S.T., and Hoffman, J.M. (1999). Acute blood flow changes and efficacy of vagus nerve stimulation in partial epilepsy. Neurology 52, 1166–1173. 7. Dorr, A.E. (2006). Effect of vagus nerve stimulation on serotonergic and noradrenergic transmission. J. Pharmacol. Exp. Ther. 318, 890–898. 8. Sitt, J.D., King, J.-R., Karoui, I.E., Rohaut, B., Faugeras, F., Gramfort, A., Cohen, L., Sigman, M., Dehaene, S., and Naccache, L. (2014). Large scale screening of neural signatures of consciousness in patients in a vegetative or minimally conscious state. Brain 137, 2258–2270. 9. Koch, C., Massimini, M., Boly, M., and Tononi, G. (2016). Neural correlates of consciousness: progress and problems. Nat. Rev. Neurosci. 17, 307–321. 10. King, J.-R., Sitt, J.D., Faugeras, F., Rohaut, B., El Karoui, I., Cohen, L., Naccache, L., and Dehaene, S. (2013). Information sharing in the brain indexes consciousness in noncommunicative patients. Curr. Biol. 23, 1914–1919.
1
Institut des Sciences Cognitives Marc Jeannerod, UMR5229, Centre National de la Recherche Scientifique (CNRS), Bron, France. 2Claude Bernard University of Lyon I, Lyon, France. 3Neurological Hospital Pierre Wertheimer, Hospices Civils de Lyon, Lyon, France. 4These authors contributed equally. *E-mail:
[email protected]
R996
Correspondence
How birds outperform humans in multi-component behavior Sara Letzner1,2,*, Onur Güntürkün2, and Christian Beste1,3,* Recent years have witnessed an astonishing flurry of studies demonstrating that some bird species show higher-order cognitive processes on par with primates [1–3]. As birds have no neocortex, cortical processing cannot be a requirement for higher order cognition [1,4]. Although birds have more neurons than expected from their small brain weights [5], their absolute neuron count is still lower compared to cortical neuron numbers of primates. How, then, is it possible that pigeons reach performance levels in, for example, abstract numerical competence and orthographic processing, that are comparable to that of macaques [6]? While the subpallium is very similar, the organization of the pallium differs tremendously between birds and mammals [1]; moreover, the avian pallium is characterized by small, extremely tightly packed neurons [5]. It is conceivable that signal processing could be faster in such a brain as a result of a higher speed of propagation of activation between neighboring assemblies, resulting in faster switch times between neighboring networks and neuronal representations of behavioral goals. This is important, as behavioral goals in real-life situations are often achieved by a series of subtasks [7,8], and especially when subtasks supersede each other and show little overlap in processing resources, neocortical (pallial) structures are involved [7,8]. We now report that pigeons are on par with humans when a task demands simultaneous processing resources; importantly, pigeons show faster responses than humans when sub-tasks are separated such that fast switches between processes are required. To test such a proposition, a behavioral procedure is needed that: can be applied similarly to
Current Biology 27, R979–R1001, September 25, 2017 © 2017 Elsevier Ltd.
birds and mammals; normalizes species-specific performance for simultaneous processing; and enables a quantitative analysis of behavioral switch speed. We used a Stop–Change task (SCT) with 15 humans and 12 pigeons (Columba livia; see Supplemental Information for details). The humans and pigeon subjects were required to perform a series of sub-tasks by stopping an ongoing response and then shifting to an alternative response (Figure 1A,B). This shift/change in responses was signaled either at the same time as the stop process with a STOP– CHANGE delay of 0 ms (SCD0), or with a short STOP–CHANGE delay of 300 ms (SCD300). In the SCD0condition sub-tasks simultaneously demand processing resources — a condition that has been shown to be mediated via the basal ganglia [9]. In the SCD300-condition, however, subtasks were separated, resulting in a lower overlap of STOP and CHANGErelated processes, which has been suggested to be strongly mediated via cortical/pallial structures [8]. Our results show that pigeons and humans did not differ in their reaction times (RTs) on trials where an ongoing response was not interrupted (GO trials) (t(25) = –0.03; p > 0.9) (Figure 1C). Regarding the SCD trials, there were longer RTs in the SCD0 than in the SCD300 condition (F(1,25) = 223.81; p < 0.001; p2 = 0.9), which is generally observed in that paradigm [8] because two response options simultaneously demand processing resources in the SCD0 condition, but less so in the SCD300 condition [8]. Importantly, there was an interaction ‘SCD condition x species’ (F(1,25) = 21.76; p < 0.001; p2 = 0.465). As expected, post-hoc tests show that there were no RT differences between humans and pigeons in the SCD0 condition (t(25) = 0.47; p > 0.6; Figure 1C), which heavily relies on basal ganglia processes [9]. This behavioral finding nicely reflects the high structural similarity of the basal ganglia between birds and mammals [1]. Importantly, in the SCD300 condition pigeons showed ~200 ms faster RTs than humans (t(25) = 3.05; p = 0.002; Figure 1C). This behavioral pattern is validated by a further experiment to support
