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Anatomic concordance in cortical preserved areas in youthful and "fit" brains
Submit responseWe read this paper with great interest. We considered of particular interest the results showing preservation of cortical thickness in specific regions that play a role as hubs of the salience and default mode networks in "superagers", in comparison with older adults with typical age-related expected decline in cognition.
As physical activity is a well know protective factor against cognitive decline, it would be interesting to know if any measure of aerobic fitness or exercise index (current or past) are at disposal to examine possible differences between those groups. We believe this would be particularly important considering our recently published data showing metabolic changes measured with FDG-PET in areas that correspond to hubs of both networks in subjects with mild cognitive impairment (MCI) after 24 weeks of aerobic training (Porto et al., 2015). Surprisingly, the areas with more important metabolic changes were the rostral anterior cingulate cortex (ACC) (decrease in resting-state metabolism) and the retrosplenial cortex (increase in resting-state metabolism). Also, physical activity decreased the intensity of the precuneus metabolic impairment in the MCI group in relation to a control group after the aerobic training. We hypothesized an increase in efficacy in the salience network induced by physical training (reflected by its decreased metabolism at rest) may influence the default mode network due to better regulation of brain networks, increasing the metabolism at rest in the precuneus, an important node of this network.
A volumetric increase in the rostral ACC was already shown after an aerobic training program in cognitively normal older adults (Colcombe et al., 2006). Interestingly, while studying the acute effects of aerobic exercise in rest brain glucose metabolism in normal young adults, other authors found a generalized metabolic decrease in all investigated areas, particularly in the dorsal ACC (Kemppainen et al., 2005). When participants were split into two groups ("trained" versus "less trained"), the trained group had a noticeable reduction in the dorsal ACC when compared to the less trained group, suggesting some type of long-term adaptive metabolic changes in the region. This is concordant with our findings. As others studies of "superagers" have been showing preservation of the ACC (Harrison et al., 2012; Rogalski et al., 2012; Gefen et al., 2015), this node of the salience network may have an important role in the relationship between physical fitness and cognitive preservation.
It is possible that neuroplasticity induced by long-term physical exercise may also influence the thickness of the cortex in "superagers", as there is an anatomical concordance between brain regions that changed after our (and others) aerobic program and those preserved in older adults with youthful memory. There might be an important link between both studies, eventually suggesting potential mechanisms between global health and the "superagers" cortical signature.
A suggestion for future studies is to take into account measures of past and current physical fitness when comparing cognition, brain structure and function in different groups of older subjects, once it is possible that better fitness is related to healthier brain aging.
References:
Colcombe SJ, Erickson KI, Scalf PE, Kim JS, Prakash R, McAuley E, Elavsky S, Marquez DX, Hu L, Kramer AF (2006) Aerobic exercise training increases brain volume in aging humans. J Gerontol A Biol Sci Med Sci 61:1166-1170.
Gefen T, Peterson M, Papastefan ST, Martersteck A, Whitney K, Rademaker A, Bigio EH, Weintraub S, Rogalski E, Mesulam M-MM, Geula C (2015) Morphometric and histologic substrates of cingulate integrity in elders with exceptional memory capacity. J Neurosci 35:1781-1791.
Harrison TM, Weintraub S, Mesulam MM, Rogalski E (2012) Superior memory and higher cortical volumes in unusually successful cognitive aging. J Int Neuropsychol Soc 18:1081-1085.
Kemppainen J, Aalto S, Fujimoto T, Kalliokoski KK, L?ngsj? J, Oikonen V, Rinne J, Nuutila P, Knuuti J (2005) High intensity exercise decreases global brain glucose uptake in humans. J Physiology 568:323-332.
Porto FH, Coutinho AM, Pinto AL, Gualano B, Duran FL, Prando S, Ono CR, Sp?ndola L, Oliveira MO, Vale PH, Nitrini R, Buchpiguel CA, Brucki SM (2015) Effects of Aerobic Training on Cognition and Brain Glucose Metabolism in Subjects with Mild Cognitive Impairment. J Alzheimer's Dis 46:747-760.
Rogalski EJ, Gefen T, Shi J, Samimi M, Bigio E, Weintraub S, Geula C, Mesulam MM (2012) Youthful Memory Capacity in Old Brains: Anatomic and Genetic Clues from the Northwestern SuperAging Project. J Cognitive Neurosci 25:29-36.
Sun FW, Stepanovic MR, Andreano J, Barrett LF, Touroutoglou A, Dickerson BC (2016) Youthful Brains in Older Adults: Preserved Neuroanatomy in the Default Mode and Salience Networks Contributes to Youthful Memory in Superaging. J Neurosci 36:9659-9668.
Conflict of Interest:
None declared
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Calpain activation in Alzheimer's disease: Comments on Saito et al.
Submit responseSaito and colleagues stated that 60% of the phenotypes observed in traditional AD mouse models are merely "artifact", based on the absence of these phenotypes in their APP knock-in mouse models carrying two or three distinct APP mutations (Saito et al., 2014). In this line, they claimed that calpain activation, p25 production and Nav1.1 reduction are all artifacts of APP and PS1 overexpression. Careful examination of the limited data presented, however, shows that this conclusion is not strongly supported. The data suffer from absence of some controls and insufficient analysis. Specific points that should be considered are highlighted below:
1. APP duplication is known to cause familial Alzheimer's disease. Similarly, individuals with Down's syndrome caused by trisomy of chromosome 21 (which contains the APP gene) also develop early-onset Alzheimer's disease. Moreover, there is evidence that individual neurons in sporadic AD patients show differences in APP gene copy number (Bushman et al., 2015). Therefore, given that APP duplication (and its overexpression) is a cause of Alzheimer's disease, it is misleading to characterize the increased calpain activity as an artifact of APP overexpression.
2. The authors suggest that the differences they observe between their APP mutant knock-in model and other transgenic models are simply due to the presence or absence of APP overexpression. This is a very limited interpretation. It is well established that AD-related pathogenic phenotypes in mouse models not only relate to APP expression and total Abeta generation, but also to Abeta oligomer formation and localization, both neuroanatomic and subcellular. These important factors have not been analyzed in this paper or the preceding report (Saito et al., 2014).
3. Increased calpain activity and marked reduction of calpastatin (Cast) have not only been shown in mouse models of Alzheimer's disease, but have also been widely reported in post-mortem brain samples of Alzheimer's disease patients (for instance, Saito et al., 1993; Rao et al., 2008; Laske et al., 2015; Kurbatskava et al., 2016). Thus, the lack of increased calpain activity in the mouse model used by Saito and colleagues calls into question the physiological relevance of their model, and whether the APP23 and other transgenic models are closer approximations of the human disease.
4. Increased p25 generation in Cast-KO mice under pathological conditions has been shown previously (Sato et al., 2011). However, in Fig. 1, Cast-KO/APPNL-F/NL-F mice do not show increased p25 signal, which should occur if as expected, it reflects calpain-dependent p35 cleavage. This raises the question of specificity and whether the authors are in fact measuring p25. The authors extensively discuss crosses with a Cast-KO mouse, but they do not show any p25 data from Cast-KO mouse brain or APP23 crossed to Cast-KO. These blots should show elevated p25 signal and would serve as positive controls.
5. The knock-in APPNL-F/NL-F mice were examined only at 2 years of age. It is possible that calpain activity is downregulated by this late age. It is also possible that calpain gene(s) are disrupted in their model. These possibilities may explain why the cross of APPNL-F/NL-F with Cast-KO did not increase p25 generation. Moreover, examination of the gel in Fig. 1 suggests that p35 may be elevated in APPNL-F/NL-F compared with the other genotypes, consistent with calpain downregulation. These control experiments should be conducted before making conclusions. To rigorously assess p25 generation, a broader range of ages should also be analyzed.
A similar concern applies to Fig 2, in which conclusions about sodium channel expression are based solely on a 2-year-old brain sample, and is generalized to suggest that a previous report of sodium channel downregulation in the J20 model was unique to that model. Furthermore, the present paper utilizes Western blotting of whole brain homogenates, a highly insensitive approach for assessing sodium channel regulation in specific interneuron subpopulations. In particular, there is no in situ analysis by immunofluorescence, electrophysiology or other methods.
6. The authors state that calpain-mediated p25 generation is an artifact caused by membrane protein overexpression such as APP or PS1, but they never address this directly. There are multiple studies demonstrating that in the absence of APP overexpression, organotypic hippocampal slices, cultured primary neurons, and even primary microglia exposed to Abeta show increased p25 generation (for instance, Zheng et al., 2005; Ma et al., 2013; Seo et al., 2014). Moreover, PS1 mutations were shown indisputably to disrupt calcium homeostasis and activate calcium-dependent enzymes (Morgan et al., 2007; Muller et al., 2011), which can increase calpain activity and subsequent p25 generation. Extending claims of artifact to PS1-related mouse models when their analysis is limited to APP models is scientifically unwarranted.
7. Finally, there is a large body of literature from many mouse models of neurological disorders that shows p25 production that is unrelated to overexpression of membrane proteins. These include models of tauopathies (Rao et al., 2014), ischemia (Meyer et al., 2014), and Parkinson's disease (Smith et al., 2006). Saito et al (2016) did not cite any of these studies. Furthermore, the statement that "overexpression of a mutant tau protein, a cytosolic protein, exhibited no effect on p25 levels (data not shown)" directly contradicts previous literature, a discrepancy that was not discussed. Based on points 6 and 7, it is misleading to characterize the p25 generation as an artifact of membrane protein overexpression.
References
Bushman DM, Kaeser GE, Siddoway B, Westra JW, Rivera RR, Rehen SK, Yung YC, Chun J. Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains. Elife 2015 doi: 10.7554/eLife.05116
Kurbatskaya K, Philips EC, Croft CL, Dentoni G, Hughes MM, Wade MA, Al-Sarraj S, Troakes C, O'Neill MJ, Perez-Nievas BG, Hanger DP, Nobel W. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain. Acta Neuropathol Commun 2016 4:34.
Laske C, Stellos K, Kempter I, Stransky E, Maetzler W, Fleming I, Randriamboavonjy V. Increased cerebrospinal fluid calpain activity and microparticle levels in Alzheimer's disease. Alzheimers Dement 2015 11(5):465-74.
Ma Y, Bao J, Zhao X, Shen H, Lv J, Ma S, Zhang X, Li Z, Wang S, Wang Q, Ji J. Activated cyclin-dependent kinase 5 promotes microglial phagocytosis of fibrillar beta-amyloid by up-regulating lipoprotein lipase expression. Mol Cell Proteomics 2013 12(10):2833-44.
Meyer DA, Torres-Altoro Ml, Tan Z, Tozzi A, Di Filippo M, DiNapoli V, Plattner F, Kansy JW, Benkovic SA, Huber JD, Miller DB, Greengard P, Calabresi P, Rosen CL, Bibb JA. Ischemic stroke injury is mediated by aberrant Cdk5. J Neurosci 2014 34(24):8259-67.
Morgan GA, Guo Q, Chan SL, Gary DS, Osborne BA, Mattson MP. Defects of immune regulation in the presenilin-1 mutant knockin mouse. Neuromolecular Med 2007 9(1):35-45.
Muller M, Cardenas C, Mei L, Cheung KH, Foskett JK. Constitutive cAMP response element binding protein (CREB) activation by Alzheimer's disease presenilin-driven inositol trisphosphate receptor (InsP3R) Ca2+ signaling. Proc Natl Acad Sci U S A 2011 109(32):13293-8.
Rao MV, Mohan PS, Peterhoff CM, Yang DS, Schmidt SD, Stavrides PH, Campbell J, Chen Y, Jiang Y, Paskevich PA, Cataldo AM, Haroutunian V, Nixon RA. Marked calpastatin (CAST) depletion in Alzheimer's disease accelerates cytoskeleton disruption and neurodegeneration: neuroprotection by CAST overexpression. J Neurosci 2008 28(47):12241-54.
Rao MV, McBrayer MK, Campbell J, Kumar A, Hashim A, Sershen H, Stavrides PH, Ohno M, Hutton M, Nixon RA. Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice. J Neurosci 2014 34(28):9222-34.
Saito K, Elce JS, Hamos JE, Nixon RA. Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration. Proc Natl Acad Sci U S A 1993 90(7):2628-32.
Saito T, Matsuba Y, Mihira N, Takano J, Nilsson P, Itohara S, Iwata N, Saido TC. Single App knock-in mouse models of Alzheimer's disease. Nat Neurosci 2014 17(5):661-3.
Sato K, Minegishi S, Takano J, Plattner F, Saito T, Asada A, Kawahara H, Iwata N, Saido TC, Hisanaga S. Calpastatin, an endogenous calpain- inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25. J Neurochem 2011 117(3):504-15.
Seo J, Giusti-Rodriguez P, Zhou Y, Rudenko A, Cho S, Ota KT, Park C, Patzke H, Madabhushi R, Pan L, Mungenast AE, Guan JS, Delalle I, Tsai LH. Activity-dependent p25 generation regulates synaptic plasticity and Abeta- induced cognitive impairment. Cell 2014 157(2):486-98.
Smith PD, Mount MP, Shree R, Callaghan S, Slack RS, Anisman H, Vincent I, Wang X, Mao Z, Park DS. Calpain-regulated p35/Cdk5 plays a central role in dopaminergic neuron death through modulation of the transcription factor myocyte enhancer factor 2. J Neurosci 2006 26(2):440-7.
Zheng YL, Kesavapany S, Gravell M, Hamilton RS, Schubert M, Amin N, Albers W, Grant P, Pant HC. A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons. EMBO J 2005 24(1):209-20.
Conflict of Interest:
None declared
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Re:The Burden of Proof Using Nanotechnology
Submit responseThis is a response to the comment submitted by Gary Sieck and colleagues entitled, The Burden of Proof using Nanotechnology.
Sieck et al., 2016 cited our study incorrectly, and focused on facts that distract from our major conclusion: the injection of a tripartite nanoconjugate comprised of a transporter (WGA-HRP), a coupler (gold nanoparticles) and a recovery-inducing drug, DPCPX, induces recovery of a hemidiaphragm paralyzed by a C2 spinal cord hemisection in rats (Minic et al., 2016). The recovery was extensive (75%) and lasted for 4 weeks after injection (the longest duration studied).
Previously we found that systemic administration of theophylline and DPCPX induces recovery in rats (Nantwi and Goshgarian, 2002), however, in humans, theophylline, not DPCPX as Sieck and colleagues suggest, induces unwanted side effects because of the ubiquitous exposure of the drugs to CNS centers. In our previous study (Tzelepis et al., 2006) 14 of 24 patients dropped out of the study because they could not tolerate theophylline. Importantly, DPCPX has not been approved for human use and it does not "induce unwanted off-target side effects" as Sieck and colleagues claim.
Furthermore, the "goal" of our study was to not "provide DPCPX at a fraction (0.1%) of the systemic dose" as suggested by these authors. This in fact, was what we measured in our study. The goal of the study was to specifically target the drug exclusively to the respiratory centers bypassing all the other CNS centers that induce side effects. The present study is a proof-of-concept study; the engineering of the theophylline-nanoconjugate is nontrivial and needs more work before publication.
We define the 'respiratory centers' as both the phrenic nucleus and the rostral ventral respiratory group (rVRG) based on the retrograde transsynaptic transport of the WGA-HRP-nanoconjugate. This was clear in our discussion. It appears that these investigators thought that 'respiratory centers' were only rVRGs. We have studied the retrograde transsynaptic transport of free conjugates of WGA (i.e. -HRP, -Alexa 488 and -Alexa 594) in 4 studies involving both acute and chronic rats using multiple modes of administration: intradiaphragmatic, intrapleural and phrenic nerve application (Moreno et al., 1992, Buttry and Goshgarian, 2014, Goshgarian and Buttry, 2014, Buttry and Goshgarian, 2015). Although we have seen transport to other CNS centers in chronically injured rats, the transport is exclusively to the phrenic nuclei and the rVRG in the acute rats (Buttry and Goshgarian, 2014). We confirmed this same result by injecting the WGA-HRP-nanoconjugate intradiaphragmatically in 3 rats in the present study. Sieck and colleagues suggest that this is insufficient data. How many more rats do we need to kill before we can establish that free conjugates of WGA have the same pattern of retrograde transsynaptic transport when the WGA is conjugated to nanodevices? Furthermore, we never stated that the recovery was mediated by targeted delivery of DPCPX specifically and exclusively to the rVRG. Since excitation of phrenic motoneurons by DPCPX may result in some recovery in addition to excitation of the rVRGs, both may be involved, as we suggested in the discussion.
Furthermore, Sieck and colleagues claim that we "failed to support [our] conclusion that intramuscular injection of the DCPPX-nanoconjugate increased respiratory premotor drive emanating from the rVRG by 75%." In addition, they state that an increase in respiratory frequency is not an effect reflecting increased premotor drive. We never stated that intramuscular injection of the DPCPX-nanoconjugate increased respiratory premotor drive emanating from the rVRG. We explained in the discussion that the increased respiratory drive may be explained by the dense expression of adenosine A1 receptors on the neurons within the central respiratory centers including the phrenic nuclei and the rVRG. Furthermore, we explained in the same paragraph that increased respiratory frequency may be a consequence of the drug action on the cells within the Botzinger complex. We explained how selective delivery of the nanoconjugate to the rVRG could impact neurons of the Botzinger complex.
The last point brought up by these investigators is notable. The release kinetics of the nanoconjugate is needed in correlative in vitro and in vivo studies to determine the pharmacokinetic profiling and to determine how much DPCPX is released and at which sites. This study was initiated several months ago in our laboratories.
References:
Buttry JL, Goshgarian HG (2014) Injection of WGA-Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity-dependent synaptic plasticity in the respiratory motor pathways. Experimental neurology 261:440-450.
Buttry JL, Goshgarian HG (2015) WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection. Journal of neuroscience methods 241:137-145.
Goshgarian HG, Buttry JL (2014) The pattern and extent of retrograde transsynaptic transport of WGA-Alexa 488 in the phrenic motor system is dependent upon the site of application. Journal of neuroscience methods 222:156-164.
Minic Z, Zhang Y, Mao G, Goshgarian HG (2016) Transporter Protein- Coupled DPCPX Nanoconjugates Induce Diaphragmatic Recovery after SCI by Blocking Adenosine A1 Receptors. The Journal of Neuroscience 36:3441-3452.
Moreno DE, Yu X-J, Goshgarian HG (1992) Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat. Experimental neurology 116:219-228.
Nantwi K, Goshgarian H (2002) Actions of specific adenosine receptor A1 and A2 agonists and antagonists in recovery of phrenic motor output following upper cervical spinal cord injury in adult rats. Clinical and experimental pharmacology and physiology 29:915-923.
Sieck, G.C, 2016, the burden of proof using nanotechnology (electronic response to Goshgarian et al.Transporter Protein-Coupled DPCPX Nanoconjugates Induce Diaphragmatic Recovery after SCI by Blocking Adenosine A1 Receptors-J. Neurosci. 2016 36:3441-3452; doi:10.1523/JNEUROSCI.2577-15.2016
Tzelepis GE, Bascom AT, Badr MS, Goshgarian HG (2006) Effects of theophylline on pulmonary function in patients with traumatic tetraplegia. The journal of spinal cord medicine 29:227.
Conflict of Interest:
None declared
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Challenging the GABA developmental sequence requires convincing data
Submit responseChallenging the GABA developmental sequence requires convincing data
Khazipov and colleagues suggest that the GABA developmental sequence is valid in slices but not in anaesthetized mice. The major limitations of this study however preclude drawing any conclusion.
Slice experiments: As neither cell attached recordings nor perforated patch of principal cells have been made the authors cannot conclude that GABA exerts excitatory or inhibitory actions. Between 2012 and 2016, Khazipov, Staley and colleagues have reported contradictory observations on GABA actions in slices. In 2012, Khazipov, Staley and colleagues suggested that GABA excitation is an artifact due to damage in slices particularly of neurons on the surface(Dzhala et al., 2012). One year later, Khazipov and colleagues suggests that GABA excitation is genuine until P3-5 but not after(Valeeva et al., 2013). In 2016, Staley and Glyskys confirmed their earlier conclusions showing that the developmental decline of (Cl-)i is independent of injury and genuine from P5 to P20 (Glykys and Staley, 2016). Now, Khazipov and colleagues suggest that excitatory actions of GABA are genuine between P2-P9 and inhibitory later, and slice damage was not considered. Given the intrinsic heterogeneity of immature neurons, relying on 7 unidentified neocortical and hippocampal neurons <P5 and 6 unidentified neurons in the P9-P15 group preclude drawing any conclusion.
In vivo experiments: i) Neither cell-attached recordings of action potentials nor measures of (Cl-)i levels from principal neurons have been made precluding any relevance to GABA excitatory or inhibitory actions; using whole cell exclusively hampers the conclusions
ii) The crux of this study is the determination by Khazipov and colleagues of the effects of channel rhodopsin stimulation of interneurons on the frequency of glutamate or GABA PSCs in pyramidal neurons. Neither the interneuron type nor the pyramidal neurons have been identified. The authors assume that the photo-activation of interneurons activates an undetermined number of interneurons (not shown) and assume that this inhibits unidentified pyramidal cells, i.e. reduce their ongoing spike frequency (not shown). They also assume that the polysynaptic reduction of PSC frequency is mediated by GABA A receptors (not tested) and ignore alternative possibilities (GABA-B receptors, peptides, hormones etc.). They also assume that channel rhodopsin actions are mediated exclusively by interneurons ignoring axons en passant that might disinhibit an intermediate population(Saunders et al., 2015).
iii) Drawing conclusions from such a small sample of unidentified cells is not straightforward. GABAergic PSC frequency was increased in 1 hippocampal unidentified neuron at P3, 2 at P4, and 1 neocortical one at P5 (Fig3H), glutamatergic EPSC frequency was reduced in 2 hippocampal neurons at P2, 2 at P4, 1 neocortical at P5 and 1 hippocampal and 1 neocortical neurons at P6 (Fig 6D). Drawing a conclusion from this heterogeneous/small population with no identification is an over interpretation notably because of the neuronal type dependence of GABA actions (Rheims et al., 2008).
iv) The shunting inhibitory actions of GABA in vitro are well demonstrated and are highly dependent on the levels of ongoing activity, hence the importance of taking that factor in account. In Figure 7B, in a majority of neurons, the ongoing frequency of EPSCs is quite similar in slices and in vivo (<circa 5Hz), inhibitory effects appear to be mediated by 5 neurons endowed with high frequency in vivo (>circa 15 Hz). Analysis of GABA inhibitory or excitatory actions necessitates a homogeneous panel of ongoing activity;
v) Introduction of a large electrode in the brain of a newborn induces local lesions leading to rise of K+, glutamate, toxins and growth factors, the effects of which cannot be ignored in contrast to slices where they are washed out;
vi) An abundant literature including by Khazipov and colleagues suggests that anesthetic agents (including isoflurane (3%) as used here, ketamine 20mg/kg here 80mg/kg were used) or urethane can produce coma, brain damage, caspase 3 activation and cell loss (Yon et al., 2005; Jevtovic-Todorovic et al., 2012; Turner et al., 2012; Sitdikova et al., 2013; Lebedeva et al., 2016). Urethane like ketamine alters calcium currents and dendritic propagation, and reduces glutamate release. Pain threshold is lower in pups than adults(Jennings and Fitzgerald, 1998) requiring repetitive administration of urethane and pain measures;
vii) Pups are heavily dependent on feeding and drinking every few hours and conclusions derived by long lasting recordings must take into account metabolic issues; at birth, pups die within 6 hours unless properly fed;
In sum, the authors mix various ages, consider a few neurons as representative of a heterogeneous age range, ignore the importance of identifying the recorded neurons, the time locked actions of GABA effects and the roles of recording conditions. The authors candidly admit that their results "do not disprove the excitatory actions of GABA in development ...at least before P3"; another unsubstantiated cutoff age. It is also difficult to understand the NKCC1/KCC2 developmental sequence or the alteration by bumetanide of the critical period of visual plasticity(Deidda et al., 2014) and the plethora of investigations showing the effects of early disturbance of high (Cl-)i levels. The thousands of neurons and wide range of preparations that confirm the sequence, show its biological relevance and provide a conceptual rationale cannot be overruled by recordings from a few unidentified neurons in anaesthetized pups (Ben-Ari, 2014). Virtually all our knowledge of cellular mechanisms is based on in vitro preparations and not to the best of my knowledge from anaesthetized animals. In vivo studies are relevant only in freely moving or un-anaesthetized rodents acting in a behavioral context and with well- controlled state of vigilance; then and only then are the ads of in vivo experiments conspicuous.
References
Ben-Ari Y (2014) The GABA excitatory/inhibitory developmental sequence: A personal journey. Neuroscience 279C:187-219.
Deidda G, Allegra M, Cerri C, Naskar S, Bony G, Zunino G, Bozzi Y, Caleo M, Cancedda L (2014) Early depolarizing GABA controls critical-period plasticity in the rat visual cortex. Nat Neurosci 18:87-96.
Dzhala V, Valeeva G, Glykys J, Khazipov R, Staley K (2012) Traumatic Alterations in GABA Signaling Disrupt Hippocampal Network Activity in the Developing Brain. J Neurosci 32:4017-4031.
Glykys J, Staley KJ (2016) Developmental Decrease of Neuronal Chloride Concentration Is Independent of Trauma in Thalamocortical Brain Slices. PLoS ONE 11:e0158012.
Jennings E, Fitzgerald M (1998) Postnatal changes in responses of rat dorsal horn cells to afferent stimulation: a fibre-induced sensitization. J Physiol (Lond) 509 ( Pt 3):859-868.
Jevtovic-Todorovic V, Boscolo A, Sanchez V, Lunardi N (2012) Anesthesia- induced developmental neurodegeneration: the role of neuronal organelles. Front Neurol 3:141.
Lebedeva YA, Zakharova AV, Sitdikova GF, Zefirov AL, Khazipov RN (2016) Ketamine-Midazolam Anesthesia Induces Total Inhibition of Cortical Activity in the Brain of Newborn Rats. Bull Exp Biol Med 161:15-19.
Rheims S, Minlebaev M, Ivanov A, Represa A, Khazipov R, Holmes GL, Ben-Ari Y, Zilberter Y (2008) Excitatory GABA in rodent developing neocortex in vitro. J Neurophysiol 100:609-619.
Saunders A, Oldenburg IA, Berezovskii VK, Johnson CA, Kingery ND, Elliott HL, Xie T, Gerfen CR, Sabatini BL (2015) A direct GABAergic output from the basal ganglia to frontal cortex. Nature 521:85-89.
Sitdikova G, Zakharov A, Janackova S, Gerasimova E, Lebedeva J, Inacio AR, Zaynutdinova D, Minlebaev M, Holmes GL, Khazipov R (2013) Isoflurane suppresses early cortical activity. Ann Clin Transl Neurol 1:15-26.
Turner CP, Gutierrez S, Liu C, Miller L, Chou J, Finucane B, Carnes A, Kim J, Shing E, Haddad T, Phillips A (2012) Strategies to defeat ketamine- induced neonatal brain injury. Neuroscience 210:384-392.
Valeeva G, Valiullina F, Khazipov R (2013) Excitatory actions of GABA in the intact neonatal rodent hippocampus in vitro. Front Cell Neurosci 7:20.
Yon J-H, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V (2005) Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135:815-827.
Conflict of Interest:
None declared
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On the problematic use of intracranial ZIP
Submit responseWe appreciate the attention Drs. Fenton and Sacktor have given to our work and their concern at reconciling their previous results using Zeta inhibitory peptide (ZIP) with ours. We need to highlight that the main point of our paper was to specifically demonstrate a troubling confound of the use of intracranial ZIP, namely, a severe disruption of neural activity, which we believe has been clearly documented in our work (LeBlancq et al., 2016). From our standpoint, the demonstration that this agent can suppress spontaneous network interactions and also generate seizure activity is troubling for prior interpretations that suggested a more specific and benign action on PKMzeta.
More generally, we would like to take this opportunity to caution against the common approach taken in neuroscience which involves jumping directly from the molecular/biochemical level of investigation to the whole behaving animal without carefully taking into consideration the intervening levels of cellular, network, and systems neuroscience. This is a not a focused criticism of the PKMzeta/ZIP story per se, but rather a more widespread criticism of the entire field.
Below we address the specific points made by Drs. Fenton and Sacktor in their comment:
1) Pastalkova et al., (2006) reported a lack of effect of ZIP on baseline evoked field potentials, specifically fEPSP slope. However, a rapid and nearly complete (i.e. lower than baseline) depression of population spikes was reported and shown in Figure 1E. Moreover, Volk et al., (2013) reported a ZIP-induced decrease in baseline evoked potential amplitude in the hippocampal slice using far less ZIP than that used in any in vivo study. In our present study, we did not test the effects of ZIP on evoked potentials so we cannot comment further. However, based on our previous work that studied the effects of intrahippocampal infusions of the translational inhibitor anisomycin (another amnestic substance that was strongly suggested to have little to no influence on neural activity - i.e., Davis and Squire, 1984), we observed a similar catastrophic loss of both spontaneous AND evoked potentials (Sharma et al, 2012). We consider it likely that we would have observed a parallel finding using ZIP in our current study had we examined evoked responses as well.
2) Although Barry et al. (2012) reported no changes in single unit hippocampal electrophysiology (other than spatial remapping) post-ZIP, we note that no data were explicitly shown for the period of time reported in our present study, i.e., within a 2 hour time window post infusion. We agree that an explicit demonstration of a behavioural effect of ZIP in the complete absence of any detrimental electrophysiological effect (most importantly including changes in local field ensemble dynamics), would go a long way in demonstrating a more specific effect of ZIP. However, in the absence of this, and in the face of our present demonstration, we still feel that our results point out a serious confound of ZIP.
3) Although it might seem difficult to reconcile the effects we report as an explanation for some prior behavioural results, notably the demonstration of ZIP-induced amnesia at longer latencies from initial training (typically one day or more), we note that very few studies of ZIP have gone beyond the one or two day limit (Kwapis and Helmstetter, 2014), which is well within the window of retrograde amnesia reported for the majority of hippocampal lesion studies (Sutherland et al, 2010). Given the recent demonstrations of the excitotoxic nature of ZIP (Hanif et al., 2012; Sadeh et al. 2015 - both studies that we unfortunately neglected to cite in our original article) we feel that the confounding problems of intracranial ZIP are compounded. Although we reported no lesions within the time frame of our experiment, we did not use specific markers of cell death to evaluate this possibility. Interestingly, and in confirmation of our own work, Sadeh et al. (2015) showed that ZIP can also induce epileptiform activity. Although the detrimental influence of this aberrant activity is disputed in their comment, we would trust that Drs. Fenton and Sacktor would agree that it still constitutes a serious confound for ZIP, since it is well known that amnestic effects can be promoted by both spontaneous and induced epileptic events. Lastly, we know of no study that explicitly pits the effects of alterations or suppression of neural activity against a more specific biochemical effect of ZIP as the primary explanation of amnesia induced by this agent. In the absence of this evidence, our findings of profound suppressive and seizure-inducing effects of ZIP are still troubling issues in the interpretation of the prior behavioural effects of ZIP. We agree with Drs. Fenton and Sacktor that this still requires definitive resolution.
4) The assertion that ZIP does not affect short-term memory does not really apply to the findings of Pastalkova et al, (2006) in which retraining was conducted outside of the 2-hour window of time reported in our study. Furthermore, this same problem applies to Shema et al., (2007), in which training was likewise conducted 2 hours post infusion. Shema et al, (2009), however, did show that ZIP infused into the insular cortex 20 minutes prior to training did not prevent a conditioned taste aversion (CTA) from being acquired as well as demonstrating that a similar ZIP infusion 1 hour following CTA training did not prevent consolidation of this memory. As are Drs. Fenton and Sacktor, we are puzzled by these results, given our clear and profound documentation of neural suppression using ZIP. Again, this is an interesting question requiring further exploration.
5) Drs. Fenton and Sacktor reflect that the (buffered) vehicle we used might be a cause of the detrimental effects on activity we report. Although we certainly agree that unbuffered acid would have (rapid) detrimental effects on neurophysiological measures (similar to those we reported for the sodium channel blocker lidocaine which were more rapid than those we found for ZIP), had this indeed been the case, we would have expected similar results for the influences of scrambled ZIP in addition to the presence of noticeable histological lesions for both ZIP and scrambled ZIP; neither of which were observed. We believe that our interpretation of a detrimental effect of ZIP on activity is a more probable and substantially less convoluted idea than the notion that PKMzeta might have a "neuroprotective effect on the potentially toxic action of intracranial injections of acetic acid".
We and many other groups are compelled by the prior behavioural results shown with ZIP. However, the demonstration of continued amnestic effects of ZIP in PKMzeta knock outs (Volk et al., 2013; Lee et al., 2013), multiple reports of the neurotoxicity of ZIP (Hanif et al., 2012, Sadeh et al., 2015), in addition to our demonstration of detrimental influences on network activity (LeBlancq et al., 2016) shake the clear interpretation of these prior data.
In conclusion, we believe that the ramifications of the suppressive and disruptive influences of ZIP as demonstrated here (LeBlancq et al., 2016) and elsewhere (Hanif et al., 2012, Sadeh et al., 2015) for prior behavioural results remains to be further explored and clarified.
References
Barry JM, Rivard B, Fox SE, Fenton AA, Sacktor TC, Muller RU (2012) Inhibition of protein kinase Mzeta disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. J Neurosci 32:13753- 13762
Davis HP, Squire LR (1984) Protein synthesis and memory: a review. Psychol Bull 96:518 -559.
Hanif W, Wang M, Sutherland RJ (2012) zeta-Pseudosubstrate Inhibitory Peptide (ZIP) is neurotoxic when infused into the hippocampus in anesthetized rat. Society for Neuroscience Conference Abstract 44.03. Society for Neuroscience.
Kwapis JL, Helmstetter FJ (2014) Does PKM(zeta) maintain memory? Brain Res Bull 105:36-45.
LeBlancq MJ, McKinney TL, and Dickson CT (2016) ZIP It: Neural Silencing Is an Additional Effect of the PKM-Zeta Inhibitor Zeta- Inhibitory Peptide J Neurosci 36:6193-6198.
Pastalkova E, Serrano P, Pinkhasova D, Wallace E, Fenton AA, Sacktor TC (2006) Storage of spatial information by the maintenance mechanism of LTP. Science 313:1141-1144.
Sadeh N, Verbitsky S, Dudai Y, and Segal M (2015) Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase M?, Is Excitotoxic to Cultured Hippocampal Neurons. J Neurosci, 35: 12404-12411.
Sharma AV, Nargang FE, Dickson CT (2012) Neurosilence: Profound Suppression of Neural Activity following Intracerebral Administration of the Protein Synthesis Inhibitor Anisomycin The Journal of Neuroscience 32:2377-2387.
Shema, R., Sacktor, T. C., Dudai, Y. (2007). Rapid Erasure of Long- Term Memory Associations in Cortex by an Inhibitor of PKMzeta, Science, 317:951-953.
Shema R., Hazvi S., Sacktor T. C., Dudai Y. (2009). Boundary conditions for the maintenance of memory by PKMzeta in neocortex. Learn Mem: 16:122-128.
Sutherland RJ, Sparks FT, Lehmann H (2010) Hippocampus and retrograde amnesia in the rat model: a modest proposal for the situation of systems consolidation. Neuropsychologia 48:2357-2369.
Conflict of Interest:
None declared
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Zeta inhibitory peptide, so what is it doing after all?
Submit responseIn their comment, Fenton and Sacktor propose that our results on ZIP and ScrZIP in cultured hippocampal neurons (Sadeh et al. 2015), contradict numerous published observations regarding specific effects of ZIP on plasticity and PKMzeta. However, their conclusion seems to overlook some published data. There are two issues that need to be dealt with separately: is PKMzeta a ???memory molecule', and is ZIP a selective blocker of PKMzeta. Our paper (Sadeh et al. 2015) does not deal with the first issue. We did not study memory, plasticity (e.g., LTP), or PKMzeta in that paper. What we did do, was test the effects of ZIP on neuronal activity in tissue culture and brain slices, effects that may or may not prove relevant to the previously published data on the postulated role of ZIP in plasticity or memory.
Fenton and Sacktor posit that our results are irrelevant to previously published data on the physiological effects of ZIP because of the high concentrations used. However, we did report an effect on network activity already at 1uM ZIP, and significant toxicity at 5uM in culture. These concentrations are 3-4 orders of magnitude below those injected into the brain in previous experiments. It has been reported (Bogard and Tavalin, 2015) that the KD of ZIP for PKMzeta is near 0.5uM, and of ScrZIP about 5uM, so one should wonder why ScrZIP does not work in vivo at 10mM if its target is indeed PKMzeta.
The timing/duration of effects of ZIP in the literature is rather confusing. In their comment, Fenton and Sacktor mention that they did not see an effect of ZIP on membrane conductance in their recent eLife paper. Yet in their supp Fig1, they record the exposure to ZIP for only 10 minutes. This might have been too short a time to see an effect, as can be seen in their extracellular recording (Figure 1A) and in our slice data.
Fenton and Sacktor further argue that with one exception (Volk et al, 2013), nobody has reported an effect of ZIP on baseline synaptic responses. Yet in their own original publication (Pastalkova et al, 2006, Figure 1E) they show a distance-dependent immediate silencing of population spikes in the hippocampus, lasting for at least 2 hours. This in fact much preceded the recent report (Leblancq et al. 2016) that ZIP can silence hippocampal activity. Notably, Leblancq et al. (2016) report that most of their ZIP-treated rats underwent an epileptic seizure, which is what could be predicted based on our in vitro data. Furthermore, in their support of a lack of effect of ZIP on hippocampal activity Fenton and Sacktor cite Barry et al, (2012). But in that study the authors recorded activity only 2 hours after ZIP, not sooner, and the activity was quite erratic.
Finally, Shema et al (2009) do not find an effect of ZIP on memory if applied right after learning, hinting at the possibility that it might not affect memory by blocking an immediate LTP-like mechanism.
Given that the ability of ZIP to block or erase long-term memory was replicated by several groups in multiple systems, it is of interest to clarify how this peptide acts. Ignoring multiple effects of ZIP that might not necessarily reinforce the hypothesis that its only target is PKMzeta, is in our view not particularly constructive in approaching the goal that we all share.
References
Barry JM, Rivard B, Fox SE, Fenton AA, Sacktor TC, Muller RU (2012). Inhibition of protein kinase Mzeta disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. J Neurosci 32(40):13753-62.
Bogard AS, Tavalin SJ (2015) Protein Kinase C (PKC)zeta Pseudosubstrate Inhibitor Peptide Promiscuously Binds PKC Family Isoforms and Disrupts Conventional PKC Targeting and Translocation. Mol Pharmacol 88(4):728-35.
LeBlancq MJ, McKinney TL, Dickson CT. (2016) ZIP It: Neural Silencing Is an Additional Effect of the PKM-Zeta Inhibitor Zeta-Inhibitory Peptide. J Neurosci. 36:6193-8.
Pastalkova E, Serrano P, Pinkhasova D, Wallace E, Fenton AA, Sacktor TC. (2006) Storage of spatial information by the maintenance mechanism of LTP. Science 313(5790):1141-4.
Sadeh N, Verbitsky S, Dudai Y, Segal M (2015) Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase Mzeta, Is Excitotoxic to Cultured Hippocampal Neurons. J Neurosci 35(36):12404-11.
Shema R, Hazvi S, Sacktor TC, Dudai Y. (2009). Boundary conditions for the maintenance of memory by PKMzeta in neocortex. Learn Mem 16(2):122-8.
Volk LJ, Bachman JL, Johnson R, Yu Y, Huganir RL. (2013) PKM-zeta is not required for hippocampal synaptic plasticity, learning and memory. Nature 493(7432):420-3.
Conflict of Interest:
None declared
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On ZIP, cellular toxicity, and the differences between the results of Sadeh et al. and other reports examining effects of ZIP on LTP and memory
Submit responseSadeh et al. highlight the need for appropriate control experiments to test for non-specific toxic effects of ZIP. Studies using ZIP have controlled for toxic effects: 1) by demonstrating the lack of effect of the equivalent dose of the scrambled peptide, which is less effective on PKMzeta than ZIP (Pastalkova et al., 2006; Lee et al., 2013; Yao et al 2013); 2) by rescuing the effect of ZIP, in which the reversal of LTP maintenance and disruption of established memory by ZIP are prevented by blocking the reversal of PKMzeta's action on AMPAR potentiation (Migues et al., 2010; Pauli et al., 2012; Evuarherhe et al., 2014); 3) by directly measuring cellular integrity using electrophysiological assays. Consequently, it is difficult to reconcile the observations of Sadeh et al. with numerous published observations.
1) Using whole-cell recordings from hippocampal slices, Tsokas et al. (2016) demonstrate no effect of 5 microM ZIP on baseline synaptic transmission (see also Wang et al., 2012), nor on PKMzeta-independent LTP induced by phorbol esters, nor on CA1 pyramidal cell membrane conductance despite the concentration of ZIP being effective at reversing PKMzeta- mediated LTP (see Tsokas et al., 2016 Fig. 1A, B, Fig. 1--figure supplement 1; https://elifesciences.org/content/5/e14846/figure1/figure- supp1).
2) Most hippocampus slice studies studying late-LTP use interface chambers for optimal slice health and maximal ZIP concentrations of 5 microM, and report no effects in baseline pathways and only effects in tetanized or facilitated pathways by measuring fEPSPs (Ling et al., 2002; Sajikumar et al., 2005; Serrano et al., 2005; Navakkode et al., 2010; Lin et al., 2012; Panaccione et al., 2013; Chen et al., 2014), and by measuring EPSCs (Li et al., 2010; Velez-Hernandez et al., 2013; Yao et al., 2013; Li et al., 2014). The only report of effects of ZIP on baseline synaptic transmission come from studies of late-LTP in submerged slices (Volk et al., 2013).
3) Pastalkova et al. (2006) report no effects of the scrambled peptide on memory (although Sadeh et al. reports toxic effects in cultured neurons of both scrambled peptide and ZIP), and demonstrate no effect of the in vivo intracranial delivery of 10 nmol/microl ZIP on baseline synaptic transmission.
4) Barry et al. (2012) demonstrate no effect of the same intracranial delivery and dose of ZIP on action potential waveforms of hippocampal pyramidal cells (place cells). The action potentials were recorded as close as 1 mm from the ZIP injection site in freely-behaving rats, demonstrating that the dose of ZIP and the method of delivery that is effective for memory disruption causes undetectable electrophysiological changes. These observations are incompatible with ZIP-induced cellular toxicity. Additional in vivo studies also provide no evidence that the effects of ZIP on memory are due to cellular toxicity (Cooke and Bear, 2010; Madronal et al., 2010; Dong et al., 2015).
It is therefore unlikely that the effects of ZIP on memory are due to cellular toxicity. The contrast with the findings of Sadeh et al. may be due to greater sensitivity of the culture system they have used as well as their use of a higher (10 microM) concentration of ZIP than has been used in prior studies to assess the role of PKMzeta in LTP.
References
Barry et al. 2012. Inhibition of protein kinase Mzeta disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. J Neurosci 32: 13753-13762.
Chen, S., et al. (2014). "Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia." Elife 3: e03896.
Cooke, S. F. and M. F. Bear (2010). "Visual experience induces long- term potentiation in the primary visual cortex." J Neurosci 30(48): 16304- 16313.
Dong, Z., et al. (2015). "Long-term potentiation decay and memory loss are mediated by AMPAR endocytosis." J Clin Invest 125(1): 234-247.
Evuarherhe O, Barker GR, Savalli G, Warburton EC, Brown MW. 2014. Early memory formation disrupted by atypical PKC inhibitor ZIP in the medial prefrontal cortex but not hippocampus. Hippocampus, 24, 934-42.
Lee, A. M., et al. (2013). "Prkcz null mice show normal learning and memory." Nature 493(7432): 416-419.
Li, W., et al. (2014). "Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain." Neurosci Lett 568: 29-34.
Li, X. Y., et al. (2010). "Alleviating neuropathic pain hypersensitivity by inhibiting PKMzeta in the anterior cingulate cortex." Science 330: 1400-1404.
Lin, Y. T., et al. (2012). "Oxytocin promotes long-term potentiation by enhancing epidermal growth factor receptor-mediated local translation of protein kinase Mzeta." J Neurosci 32: 15476-15488.
Ling, D. S., et al. (2002). "Protein kinase Mzeta is necessary and sufficient for LTP maintenance." Nat Neurosci 5: 295-296.
Madronal, N., et al. (2010). "PKMzeta inhibition reverses learning- induced increases in hippocampal synaptic strength and memory during trace eyeblink conditioning." PLoS One 5: e10400.
Migues PV, Hardt O, Wu DC, Gamache K, Sacktor TC, Wang YT, Nader K. 2010. PKM? maintains memories by regulating GluR2-dependent AMPA receptor trafficking. Nat Neurosci, 13, 630-4.
Navakkode, S., et al. (2010). "Protein kinase Mzeta is essential for the induction and maintenance of dopamine-induced long-term potentiation in apical CA1 dendrites." Learn Mem 17: 605-611.
Panaccione, I., et al. (2013). "Constitutively active group I mGlu receptors and PKMzeta regulate synaptic transmission in developing perirhinal cortex." Neuropharmacology 66: 143-150.
Pastalkova, E., et al. (2006). "Storage of spatial information by the maintenance mechanism of LTP." Science 313(5790): 1141-1144.
Pauli WM, Clark AD, Guenther HJ, O'reilly RC, Rudy JW. 2012. Inhibiting PKMzeta reveals dorsal lateral and dorsal medial striatum store the different memories needed to support adaptive behavior. Learn Mem, 19, 307-14.
Sajikumar, S., et al. (2005). "Synaptic tagging and cross-tagging: the role of protein kinase Mzeta in maintaining long-term potentiation but not long-term depression." J Neurosci 25: 5750-5756.
Serrano, P., et al. (2005). "Persistent phosphorylation by protein kinase Mzeta maintains late-phase long-term potentiation." J Neurosci 25: 1979-1984.
Tsokas et. al. 2016. Compensation for PKMzeta in long-term potentiation and spatial long-term memory in mutant mice. eLife 5:e14846.
Velez-Hernandez, M. E., et al. (2013). "Inhibition of Protein kinase Mzeta (PKMzeta) in the mesolimbic system alters cocaine sensitization in rats." J Drug Alcohol Res 2: 235669.
Wang, J., et al. (2012). "Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase M synthesis and induce immediate preconditioning protection of rat hippocampal slices." J Physiol 590: 4093-4107.
Yao, Y., et al. (2013). "Matching biochemical and functional efficacies confirm ZIP as a potent competitive inhibitor of PKMzeta in neurons." Neuropharmacology 64: 37-44.
Conflict of Interest:
None declared
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On ZIP, neuronal silencing, and the differences between the results of LeBlancq et al. and other reports examining effects of ZIP on memory
Submit responseIt is difficult to reconcile the data reported in LeBlancq et al. with a large number of published observations.
1) In the urethane-anesthetized rat, Pastalkova et al. (2006) observed that ZIP has no effect on baseline population postsynaptic responses (Fig. 1D, and supplemental Fig. S2). After inducing LTP, ZIP depotentiated postsynaptic responses, which returned to baseline values and stabilized. This was measured by both the field excitatory postsynaptic potential (fEPSP; Fig. 1C) and population (action potential) spike (PS; supplemental Fig. S1). After infusing ZIP, stable fEPSPs and PS responses depotentiated to baseline could not have been observed if ZIP had caused neuronal silencing or seizures.
2) In freely-behaving rats, Barry et al. (2012) observed that ZIP had no effects on action potential waveforms and local field potentials 2 h afterwards. Unpublished recordings were made 10-20 min after ZIP infusion, and we observed no evidence of seizure or silencing due to ZIP.
3) The LeBlancq et al. authors assert that seizure and/or temporary neuronal silencing by ZIP is sufficient to explain the amnesia for various forms of long-term memory that have been observed following ZIP infusion. But this assertion is contrary to a large body of evidence. In experiments to test its effect on established long-term memory, the peptide is infused at least 1 day after training. Seizures, the neuronal silencing induced by short-lasting lidocaine (~30 min), the longer-lasting AMPA/kainate receptor antagonist LY326325 (> 6 h), or tetrodotoxin (~12 h), are all known to effectively impair learning, the initial cellular consolidation of memory lasting a few hours, and memory retrieval. But in contrast to ZIP, these treatments do not impair the storage of memories that have had a period of 24 h or longer to consolidate after memory activation. This is demonstrated by intact retrieval of hippocampus-dependent memory, so long as the retention test is sufficiently long (> 5 h) after the seizure or neural inactivation (Fenton et al., 1994; 1995; Jezek et al., 2002; Riedel et al., 1999). Furthermore, place cell firing and spatial performance are only altered after days of repeated seizures (Lin et al., 2009; 2012). Thus, even if ZIP caused seizures or neuronal silencing (neither of which we have observed during our in vivo recordings), experiments that investigate the effect of ZIP on memory persistence infuse ZIP 1 to 30 days after training, when seizure and neuronal silencing are ineffective on memory persistence.
4) In contrast to effects on long-term memory persistence, ZIP does not affect short-term memories or initial consolidation of short-term into long-term memory (Pastalkova et al, 2006; Shema et al., 2007, 2009), precisely the forms of memory that are disrupted by neuronal silencing (Riedel, et al., 1999).
In conclusion, we do not know the source of differences between experiments that show no neuronal silencing and those of LeBlancq et al. that do, but we and others typically inject ZIP in phosphate buffered saline (PBS). LeBlancq et al. injected ZIP, PBS, and acetic acid up to 14% (v:v in PBS). It would be useful to determine whether PKMzeta has a neuroprotective effect on the potentially toxic action of intracranial injections of acetic acid.
References
Fenton, A.A., and Bures, J. (1994). Interhippocampal transfer of place navigation monocularly acquired by rats during unilateral functional ablation of the dorsal hippocampus and visual cortex with lidocaine. Neuroscience 58, 481-491.
Fenton, A.A., Arolfo, M.P., Nerad, L., and Bures, J. (1995). Interhippocampal synthesis of lateralized place navigation engrams. Hippocampus 5, 16-24.
Jezek, K., M. Wesierska, et al. (2002). "Hippocampus-dependent retrieval and hippocampus-independent extinction of place avoidance navigation, and stress-induced out-of-context activation of a memory revealed by reversible lesion experiments in rats." Physiol Res 51 Suppl 1: S35-47.
Lin, H., Holmes, G. L., Kubie, J. L., Muller, R. U. (2009). Recurrent seizures induce a reversible impairment in a spatial hidden goal task. Hippocampus 19: 817-827.
Lin, H., Hangya, B., Fox, S. E., Muller, R. U. (2012). Repetitive convulsant-induced seizures reduce the number but not precision of hippocampal place cells. J Neurosci 32: 4163-4178.
Riedel, G.Riedel, G., Micheau, J., Lam, A. G., Roloff, E. L., Martin, S. J., Bridge, H., de Hoz, L., Poeschel, B., McCulloch, J., Morris, R. G.(1999). Reversible neural inactivation reveals hippocampal participation in several memory processes. Nat Neurosci 2: 898-905.
Shema, R., Sacktor, T. C., Dudai, Y. (2007). Rapid Erasure of Long- Term Memory Associations in Cortex by an Inhibitor of PKMzeta, Science, 317:951-953
Shema R., Hazvi S., Sacktor T. C., Dudai Y. (2009). Boundary conditions for the maintenance of memory by PKMzeta in neocortex. Learn. Mem.: 16:122-128.
Tsokas et al. (2016). "Compensation for PKMzeta in long-term potentiation and spatial long-term memory in mutant mice." eLife 2016;5:e14846
Conflict of Interest:
None declared
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How about high-frequency, periodic excitation instead of noise?
Submit responseCongratulations on the nice paper!
Can you perhaps rerun the experiments with high-frequency triangle waves instead of noise? The best is to use a frequency similar to that of the high- frequency end of your noise bandwidth.
According to the results of my calculations, which I presented in the public debate session about Stochastic resonance at the Fluctuation and Noise (FaN) Symposium in Florence (2007), using triangle waves instead of noise would produce the best signal-to-noise ratio at the output of a threshold stochastic resonator and would not result in any stochasticity in the output signal.
Laszlo Kish (formerly L.B. Kiss) http://engineering.tamu.edu/electrical/people/lkish
Conflict of Interest:
None declared
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The Rich Club of the Avian Brain
Submit responseThe authors write "So far, the rich-club has been conceptualized as a large-scale brain network phenomenon in adult humans (van den Heuvel and Sporns, 2011), human newborns (Ball et al., 2014), macaque monkey (Harriger et al., 2012), the nematode worm Caenorhabditis elegans (Towlson et al., 2013), and mouse hippocampus (Schroeter et al., 2015)."
This list should have included the pigeon (Shanahan, et al., 2014):
Shanahan, M., Bingman, V., Shimizu, T., Wild, M. & Gunturkun, O., Large-Scale Network Organisation in the Avian Forebrain: A Connectivity Matrix and Theoretical Analysis, Frontiers in Computational Neuroscience, vol. 7 (2013), article 89.
http://journal.frontiersin.org/article/10.3389/fncom.2013.00089/full
In particular, see Figure 8 and the relevant text.
Conflict of Interest:
None declared
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