Challenging 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 conclud...
Challenging 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
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None declared