Beware of your Cre‐Ation: <i>lacZ</i> expression impairs neuronal integrity and hippocampus‐dependent memory

Reichel, Judith M.; Bedenk, Benedikt T.; Gassen, Nils C.; Hafner, Kathrin; Bura, S. Andreea; Almeida-Corrêa, Suellen; Genewsky, Andreas; Dedic, Nina; Giesert, Florian; Agarwal, Amit; Nave, Klaus Armin; Rein, Theo; Czisch, Michael; Deussing, Jan M.; Wotjak, Carsten T.

doi:10.1002/hipo.22601

Cited by 7 publications

(3 citation statements)

References 76 publications

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“…Golgi staining was performed as previously described (Giusti et al, 2014; Reichel et al, 2016), and to shift the staining towards glia cells instead of neurons, we adapted a modified protocol described by Ranjan and Mallick (Ranjan and Mallick, 2012). Mice were sacrificed under anesthesia, the brains carefully dissected out and briefly rinsed in dH2O, and the brains were incubated in 4% PFA-PBS at room temperature for 72 h. Subsequently the protocol of the FD Rapid GolgiStain™ Kit (FD Neurotechnologies, INC) was followed.…”

Section: Star Methodsmentioning

confidence: 99%

Astrocytic Process Plasticity and IKKβ/NF-κB in Central Control of Blood Glucose, Blood Pressure, and Body Weight

et al. 2017

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Summary Central regulation of metabolic physiology is mediated critically through neuronal functions; however, if astrocytes are also essential remains unclear. Here we show that the high-order processes of astrocytes in the mediobasal hypothalamus displayed shortening in fasting while elongation in fed status. Chronic overnutrition and astrocytic IKKβ/NF-κB upregulation similarly impaired astrocytic plasticity leading to sustained shortening of high-order processes. In physiology, astrocytic IKKβ/NF-κB upregulation resulted in early-onset effects including glucose intolerance and blood pressure rise and late-onset effects including body weight and fat gain. Appropriate inhibition in astrocytic IKKβ/NF-κB protected against chronic overnutrition from impairing astrocytic plasticity and these physiological functions. Mechanistically, astrocytic regulation of hypothalamic extracellular GABA level and therefore BDNF expression were found partly accountable. Hence, astrocytic process plasticity and IKKβ/NF-κB play significant roles in central control of blood glucose, blood pressure and body weight as well as the central induction of these physiological disorders leading to disease.

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Section: Star Methodsmentioning

confidence: 99%

Astrocytic Process Plasticity and IKKβ/NF-κB in Central Control of Blood Glucose, Blood Pressure, and Body Weight

et al. 2017

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“…Our focus has been on lacZ expression in DRG neurons; one previous study has investigated expression in cortical neurons and raised concerns regarding the use of LacZ reporters ( Reichel et al, 2016 ). The authors found that lacZ expression in cortical glutamatergic neurons caused severe morphological and behavioural impairments such as decreased hippocampal volume, reduced dendritic branching in hippocampal neurons, and deficits in hippocampus-dependent memory.…”

Section: Discussionmentioning

confidence: 99%

Studying IndependentKcna6Knock-out Mice Reveals Toxicity of Exogenous LacZ to Central Nociceptor Terminals and Differential Effects of Kv1.6 on Acute and Neuropathic Pain Sensation

et al. 2021

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The potassium channel Kv1.6 has recently been implicated as a major modulatory channel subunit expressed in primary nociceptors. Furthermore, its expression at juxtaparanodes (JXP) of myelinated primary afferents is induced following traumatic nerve injury as part of an endogenous mechanism to reduce hyperexcitability and pain-related hypersensitivity. In this study we compared two mouse models of constitutive Kv1.6 knock-out achieved by different methods: traditional gene trap via homologous recombination, and CRISPR-mediated excision. Both Kv1.6 knock-out mouse lines exhibited an unexpected reduction in sensitivity to noxious heat stimuli, to differing extents: the Kv1.6 mice produced via gene trap had a far more significant hyposensitivity. These mice (Kcna6 lacZ ) expressed the bacterial reporter enzyme LacZ in place of Kv1.6 as a result of the gene trap mechanism and we found that their central primary afferent presynaptic terminals developed a striking neurodegenerative phenotype involving accumulation of lipid species, development of 'meganeurites' and impaired transmission to dorsal horn wide dynamic range (WDR) neurons. The anatomical defects were absent in CRISPR-mediated Kv1.6 knock-out mice (Kcna6 -/-) but were present in a third mouse model expressing exogenous LacZ in nociceptors under the control of a Nav1.8-promoted Cre recombinase. LacZ reporter enzymes are thus intrinsically neurotoxic to sensory neurons and may induce pathological defects in transgenic mice, which has confounding implications for the interpretation of gene knock-outs using lacZ. Nonetheless, in Kcna6 -/mice not affected by LacZ, we demonstrated a significant role for Kv1.6 regulating acute noxious thermal sensitivity, and both mechanical and thermal pain-related hypersensitivity after nerve injury. Significance Statement 4In recent decades the expansion of technologies to experimentally manipulate the rodent genome has contributed significantly to the field of neuroscience. While introduction of enzymatic or fluorescent reporter proteins to label neuronal populations is now commonplace, often potential toxicity effects are not fully considered. We show a role of Kv1.6 in acute and neuropathic pain states through analysis of two mouse models lacking Kv1.6 potassium channels, one with additional expression of LacZ and one without. We show that LacZ reporter enzymes induce unintended defects in sensory neurons, with an impact on behavioural data outcomes. To summarise we highlight the importance of: Kv1.6 in recovery of normal sensory function following nerve injury, and careful interpretation of data from LacZ reporter models.

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“…During the sampling phase of NOR, the di/di-AVP animals spent less time investigating the novel object than the +/+ rats (Figure 6A, F(2,27) = 3.462, p = 0.046). However, even this lower level (40-55 s) should have been enough to remember the object [29]. During the choice phase, a memory deficit of the di/di rats was revealed by the NOR test (Figure 6B, difference between old and new objects: +/+: p = 0.047, di/di: p = 0.954, di/di-AVP: p = 0.224).…”

Section: Avp-aav Rescue In the Son Had No Effect On Nor But Altered The Social Behaviour (Series 1 And 2)mentioning

confidence: 95%

Rescue of Vasopressin Synthesis in Magnocellular Neurons of the Supraoptic Nucleus Normalises Acute Stress-Induced Adrenocorticotropin Secretion and Unmasks an Effect on Social Behaviour in Male Vasopressin-Deficient Brattleboro Rats

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et al. 2022

IJMS

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The relevance of vasopressin (AVP) of magnocellular origin to the regulation of the endocrine stress axis and related behaviour is still under discussion. We aimed to obtain deeper insight into this process. To rescue magnocellular AVP synthesis, a vasopressin-containing adeno-associated virus vector (AVP-AAV) was injected into the supraoptic nucleus (SON) of AVP-deficient Brattleboro rats (di/di). We compared +/+, di/di, and AVP-AAV treated di/di male rats. The AVP-AAV treatment rescued the AVP synthesis in the SON both morphologically and functionally. It also rescued the peak of adrenocorticotropin release triggered by immune and metabolic challenges without affecting corticosterone levels. The elevated corticotropin-releasing hormone receptor 1 mRNA levels in the anterior pituitary of di/di-rats were diminished by the AVP-AAV-treatment. The altered c-Fos synthesis in di/di-rats in response to a metabolic stressor was normalised by AVP-AAV in both the SON and medial amygdala (MeA), but not in the central and basolateral amygdala or lateral hypothalamus. In vitro electrophysiological recordings showed an AVP-induced inhibition of MeA neurons that was prevented by picrotoxin administration, supporting the possible regulatory role of AVP originating in the SON. A memory deficit in the novel object recognition test seen in di/di animals remained unaffected by AVP-AAV treatment. Interestingly, although di/di rats show intact social investigation and aggression, the SON AVP-AAV treatment resulted in an alteration of these social behaviours. AVP released from the magnocellular SON neurons may stimulate adrenocorticotropin secretion in response to defined stressors and might participate in the fine-tuning of social behaviour with a possible contribution from the MeA.

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Beware of your Cre‐Ation: lacZ expression impairs neuronal integrity and hippocampus‐dependent memory

Cited by 7 publications

References 76 publications

Astrocytic Process Plasticity and IKKβ/NF-κB in Central Control of Blood Glucose, Blood Pressure, and Body Weight

Astrocytic Process Plasticity and IKKβ/NF-κB in Central Control of Blood Glucose, Blood Pressure, and Body Weight

Studying IndependentKcna6Knock-out Mice Reveals Toxicity of Exogenous LacZ to Central Nociceptor Terminals and Differential Effects of Kv1.6 on Acute and Neuropathic Pain Sensation

Rescue of Vasopressin Synthesis in Magnocellular Neurons of the Supraoptic Nucleus Normalises Acute Stress-Induced Adrenocorticotropin Secretion and Unmasks an Effect on Social Behaviour in Male Vasopressin-Deficient Brattleboro Rats

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