A postnatal peak in microglial development in the mouse hippocampus is correlated with heightened sensitivity to seizure triggers

“To examine changes in the number and size of microglia during development and after FS or KA-SE, six sections spanning the extent of the dorsal hippocampus were selected from each animal (n = 3–4 animals/group).”

“Additionally, emerging evidence for the integral role played by microglia in developmental synapse maturation and elimination suggests that they not only are present, but are functionally active by this time (Stevens et al. 2007; Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012).”

“Additionally, emerging evidence for the integral role played by microglia in developmental synapse maturation and elimination suggests that they not only are present, but are functionally active by this time (Stevens et al. 2007; Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012).”

“In addition, febrile seizures (FS) occur predominantly between three months and six years of age (Stafstrom 2002), and several epilepsy syndromes resolve after childhood (Brorson and Wranne 1987).”

“Infectious and autoimmune diseases are often accompanied by recurrent seizures (Dalmau et al. 2008; Kim et al. 2009).”

“Additionally, emerging evidence for the integral role played by microglia in developmental synapse maturation and elimination suggests that they not only are present, but are functionally active by this time (Stevens et al. 2007; Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012).”

“The fractalkine receptor in these mice has been replaced by a green fluorescent protein (GFP) reporter gene by targeted deletion via homologous recombination in embryonic stem cells (Jung et al. 2000; Davalos et al. 2005).”

“Kainic acid-induced status epilepticus susceptibility Systemic injection of KA in rodents induces limbic seizures which originate in the CA3 region of the hippocampus (Nadler 1981; Ben-Ari 1985).”

“Expression of Tnf-a and Il-1b and inducible nitric oxide synthase, Nos2 are known to be upregulated by activated microglia (Hanisch 2002; Smith et al. 2012; Bechade et al. 2014).”

“Microglia are now known to be key mediators of synapse remodeling and elimination (Wake et al. 2009; Paolicelli et al. 2011; Tremblay et al. 2011).”

“Additionally, emerging evidence for the integral role played by microglia in developmental synapse maturation and elimination suggests that they not only are present, but are functionally active by this time (Stevens et al. 2007; Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012).”

“Microglia are now known to be key mediators of synapse remodeling and elimination (Wake et al. 2009; Paolicelli et al. 2011; Tremblay et al. 2011).”

“…time of dynamic synaptic pruning by microglia, it makes sense that microglia at P10–P15 are present at high densities in the hippocampus, have achieved a mature phenotype (Perry et al. 1985) and morphology, and would be especially sensitive to activation during this period (Bilbo and Schwarz 2009).”

“403 (9 of 14) modulate neuronal activity (Tremblay 2011) indirectly via influences on astrocytes (Pascual et al. 2012).”

“Administration of IL-1b prior to KA increases the time spent in seizures via an NMDA receptor-dependent mechanism (Vezzani et al. 1999).”

“IBA1, also known as Aif1, has been shown to preferentially label activated over resting microglia (Postler et al. 2000; Ito et al. 2001; Schwab et al. 2001).”

“…to hyperthermia- or hypoxia-induced seizures during the first two weeks of life (Jensen et al. 1998; Chen et al. 1999; Jensen and Baram 2000; Rakhade and Jensen 2009), and that age-dependent differences exist in microglial activation following KA-induced seizures (Rizzi et al. 2003; Ravizza…”

“Rather than improve behavioral deficits associated with epilepsy or modify disease progression, AEDs themselves can cause cognitive, behavioral, and mood-altering side effects (Kwan and Brodie 2000; Temkin 2001).”

“Additionally, mice engineered to overexpress IL-1ra in astrocytes have decreased seizure susceptibility (Vezzani et al. 2000).”

“Cells were isolated and stained as previously described (Bailey et al. 2007).”

“Pro and anti-inflammatory molecules are upregulated in the brain at the sites of seizure initiation and propagation and may influence seizure susceptibility (Murashima et al. 2008; Fukuda et al. 2009; Galic et al. 2012; Choy et al. 2014; Vezzani 2014).”

“Reduction of the biologically-active form of IL-1b by interleukin-converting enzyme significantly reduces seizure onset and duration (Ravizza et al. 2006), and intracerebral injection of IL-1 receptor antagonist (ra) produces an anticonvulsant effect (Vezzani et al. 2002).”

“Comparable to what has been observed in earlier studies with rats (Albala et al. 1984), the dose of KA sufficient to produce SE increased with age.”

“Moreover, there is evidence that activated microglia, through the integrin CD11b in cooperation with the immunoreceptor DAP12, trigger developmental apoptosis of hippocampal neurons via the production of superoxide ions (Wakselman et al. 2008).”

“Previous studies have established that immature rodents are highly susceptible to hyperthermia- or hypoxia-induced seizures during the first two weeks of life (Jensen et al. 1998; Chen et al. 1999; Jensen and Baram 2000; Rakhade and Jensen 2009), and that age-dependent differences exist in microglial activation follow…”

“Infectious and autoimmune diseases are often accompanied by recurrent seizures (Dalmau et al. 2008; Kim et al. 2009).”

“IL-1b, for instance, is rapidly upregulated in microglia after the induction of acute seizures (Vezzani et al. 2010; Jarvela et al. 2011), and receptors for IL-1b are present at high density in the hippocampus, specifically the dentate gyrus (Takao et al. 1990; Ban et al. 1991).”

“Furthermore, microglia – the resident innate immune cells of the central nervous system – are markedly activated in the brains of individuals with chronic intractable epilepsy and in animal models of seizures (Beach et al. 1995; Yang et al. 2000; Drage et al. 2002; Avignone et al. 2008; Choi et al. 2009; Somera-Molina…”

“IL-1b, for instance, is rapidly upregulated in microglia after the induction of acute seizures (Vezzani et al. 2010; Jarvela et al. 2011), and receptors for IL-1b are present at high density in the hippocampus, specifically the dentate gyrus (Takao et al. 1990; Ban et al. 1991).”

“…seizures during the first two weeks of life (Jensen et al. 1998; Chen et al. 1999; Jensen and Baram 2000; Rakhade and Jensen 2009), and that age-dependent differences exist in microglial activation following KA-induced seizures (Rizzi et al. 2003; Ravizza et al. 2005; Jarvela et al. 2011).”

“Received: 7 January 2015; Revised: 30 July 2015; Accepted: 2 September 2015 Brain and Behavior, 2015; 5(12), e00403, doi: 10.1002/brb3.”

“They are also the only cells in the CNS known to express the C3 receptor (Gasque et al. 1998), a complement factor of the classical activation cascade that is implicated, along with C1q, in a mechanism of synapse elimination (Stevens et al. 2007).”

“403 (3 of 14) was also amplified to serve as an internal control using the primers as follows: 18S rRNA, forward: 50-CGGCTAC CACATCCAAGGAA-30, reverse: 50-AGCCGCGGTAATT CCAGC-30(Hazarika et al. 2007).”

“Pro and anti-inflammatory molecules are upregulated in the brain at the sites of seizure initiation and propagation and may influence seizure susceptibility (Murashima et al. 2008; Fukuda et al. 2009; Galic et al. 2012; Choy et al. 2014; Vezzani 2014).”

“403 (1 of 14) microglia into mature forms appears to be fully complete by the third week of life (Wu et al. 1992; Streit and Xue 2009).”

“…seizures during the first two weeks of life (Jensen et al. 1998; Chen et al. 1999; Jensen and Baram 2000; Rakhade and Jensen 2009), and that age-dependent differences exist in microglial activation following KA-induced seizures (Rizzi et al. 2003; Ravizza et al. 2005; Jarvela et al. 2011).”

“Furthermore, microglia – the resident innate immune cells of the central nervous system – are markedly activated in the brains of individuals with chronic intractable epilepsy and in animal models of seizures (Beach et al. 1995; Yang et al. 2000; Drage et al. 2002; Avignone et al. 2008; Choi et al. 2009; Somera-Molina…”

“Previous studies have established that immature rodents are highly susceptible to hyperthermia- or hypoxia-induced seizures during the first two weeks of life (Jensen et al. 1998; Chen et al. 1999; Jensen and Baram 2000; Rakhade and Jensen 2009), and that age-dependent differences exist in microglial activation follow…”

“Furthermore, microglia – the resident innate immune cells of the central nervous system – are markedly activated in the brains of individuals with chronic intractable epilepsy and in animal models of seizures (Beach et al. 1995; Yang et al. 2000; Drage et al. 2002; Avignone et al. 2008; Choi et al. 2009; Somera-Molina…”

“IBA1, also known as Aif1, has been shown to preferentially label activated over resting microglia (Postler et al. 2000; Ito et al. 2001; Schwab et al. 2001).”

“Seizure susceptibility and seizure-induced microglial activation were assessed in response to febrile seizures (lipopolysaccharide followed by hyperthermia) and kainic acid-induced status epilepticus.”

“Seizure susceptibility and seizure-induced microglial activation were assessed in response to febrile seizures (lipopolysaccharide followed by hyperthermia) and kainic acid-induced status epilepticus.”

“Received: 7 January 2015; Revised: 30 July 2015; Accepted: 2 September 2015 Brain and Behavior, 2015; 5(12), e00403, doi: 10.1002/brb3.”

“Brain and Behavior published by Wiley Periodicals, Inc. age-dependent behavioral and microglial response to Kainic acid-induced status epilepticus (KA-SE), seizures were induced via i.p. injection of KA (#K-1013, A.G. Scientific, Inc., San Diego, CA) as previously described (Hu et al. 1998).”

“IBA1, also known as Aif1, has been shown to preferentially label activated over resting microglia (Postler et al. 2000; Ito et al. 2001; Schwab et al. 2001).”

“Furthermore, microglia – the resident innate immune cells of the central nervous system – are markedly activated in the brains of individuals with chronic intractable epilepsy and in animal models of seizures (Beach et al. 1995; Yang et al. 2000; Drage et al. 2002; Avignone et al. 2008; Choi et al. 2009; Somera-Molina…”

“These time points were chosen because previous work showed cytokine gene induction after FS to be immediate and shortlived, with blood levels of IL-1b, IL-6, and TNF-a peaking within 2 h and declining to baseline levels as early as 4 h after FS (Eun et al. 2015).”

“Our laboratory has developed a new experimental model of FS by combining lipopolysaccharide (LPS), a Toll-like Receptor 4 (TLR4) ligand, with hyperthermia-induced seizures in immature mice (Eun et al. 2015; Radzicki et al. 2013).”

“IL-1b, for instance, is rapidly upregulated in microglia after the induction of acute seizures (Vezzani et al. 2010; Jarvela et al. 2011), and receptors for IL-1b are present at high density in the hippocampus, specifically the dentate gyrus (Takao et al.”

“Pro and anti-inflammatory molecules are upregulated in the brain at the sites of seizure initiation and propagation and may influence seizure susceptibility (Murashima et al. 2008; Fukuda et al. 2009; Galic et al. 2012; Choy et al. 2014; Vezzani 2014).”

“…of Tnf-a and Il-1b and inducible nitric oxide synthase, Nos2 are known to be upregulated by activated microglia (Hanisch 2002; Smith et al. 2012; Bechade et al. 2014). mRNA levels for each of the genes were significantly increased compared to untreated controls both immediately following and 4…”

“Specifically, microglia express neurotransmitter receptors (Pocock and Kettenmann 2007; Hung et al. 2010) and Figure 4.”

“Pro and anti-inflammatory molecules are upregulated in the brain at the sites of seizure initiation and propagation and may influence seizure susceptibility (Murashima et al. 2008; Fukuda et al. 2009; Galic et al. 2012; Choy et al. 2014; Vezzani 2014).”

“Pro and anti-inflammatory molecules are upregulated in the brain at the sites of seizure initiation and propagation and may influence seizure susceptibility (Murashima et al. 2008; Fukuda et al. 2009; Galic et al. 2012; Choy et al. 2014; Vezzani 2014).”

“IL-1b, for instance, is rapidly upregulated in microglia after the induction of acute seizures (Vezzani et al. 2010; Jarvela et al. 2011), and receptors for IL-1b are present at high density in the hippocampus, specifically the dentate gyrus (Takao et al.”

“In humans, CD11c is a panmyeloid cell marker expressed by monocytes, macrophages, neutrophils, myeloid DCs, and microglia, much like its counterpart CD11b, the panmyeloid cell marker in mice (Cobbold and Waldmann, 1987; Kim et al., 2015).”

“Primitive microglia often display round structures in the underdeveloped processes, which present a beaded shape, consistent with the presence of engulfed material in their interior (Kim et al., 2015).”

“The expression pattern of Cx3cr1-driven GFP reporter is specific to microglia over the entire period of postnatal brain development and appears to be maintained even following an excitatory injury (Kim et al., 2015).”

“MOR Expression in CX3cr1-eGFP Microglia MOR Expression in Brain and Spinal Cord The Figures 10, 11 show that Cx3cr1-eGFP-positive cells display the typical microglia morphology with no neuronal, astrocytic or lymphocytic shapes, confirming previous findings on colocalization of CX3CR1-eGFP with the CD11b or Iba1 micro…”

“Second, while the CX3CR1 transgenic model is a standard tool to study microglial morphology in in vivo [49, 50] or ex vivo experiments [51, 52], we performed the same experiments based on Iba-1 expression in C57BL/6 mice Fig.”

“These critical periods overlap with a crucial window of microglial activity, when >95% of microglia are generated (Alliot et al. 1999), with density peaking (Kim et al. 2015; Nikodemova et al. 2015) and maturation occurring by the second postnatal week (Bennett et al. 2016).”

“Furthermore, TNF-α and IL-1β are found to be increased in the kainic acid-induced epilepsy model, and seizure susceptibility correlates with microglia development in the hippocampus [120].”

“However, microglial numbers peak in the second week of post-natal development (Kim et al., 2015; Nikodemova et al., 2015) and are trimmed by the third week (Nikodemova et al., 2015; Paolicelli et al., 2011), and although astrocytes have not been documented to be instrumental in the reduction of…”

“Anti-inflammatory therapy targeting excessive production of inflammatory mediators by activated microglia, therefore, may be an effective agespecific therapeutic strategy to minimize neuronal dysfunction and prevent increases in susceptibility to subsequent seizures in developing animals.(48) Figure 1.”

“Microglia proliferate until the fourteenth post-natal day in rodents, and after having reached the peak of proliferation, the number of microglia slowly decreases to adult levels (I. Kim et al., 2015).”

“The expression levels of microglial cytokines like inflammatory cytokines such as TNF-α and IL-1β were elevated 4 h after FSs (Kim et al., 2015).”

“Maternal separation elicits neuroinflammatory alterations in the hippocampus and prefrontal cortex of P15 WT and 5xFAD mice We first wondered whether maternal separation (MS) could induce any immediate neuroinflammatory alterations that we could detect.”

“Conflicting experimental results suggest that microglia either promote seizures (Abraham et al., 2012; Di Nunzio et al., 2021; Eun et al., 2015; Kim et al., 2015) or dampen neuronal hyperactivity (Cserép et al., 2020; Kato et al., 2016; Li et al., 2012; Merlini et al., 2021) and perform…”

“Postnatal development of microglia correlates closely with seizure susceptibility (26).”

“Activated microglia undergo quantitative and morphological changes, and morphological changes include mainly the area of the microglia cell somata and the number and length of foot processes [16, 17].”

“From rodent studies, it is known that their numbers contract in postnatal week 3 and subsequently stabilize to adult levels after week 4 [145, 146].”

“The P14 peak has been associated with increased seizure susceptibility [11].”

“Microglia are virtually absent from the mouse hippocampus at birth, but the numbers of these cells increase markedly between 5 and 10 d of age and peak at 15 d of age (29).”

“Interestingly, as found in this study and several others (Hoshiko et al., 2012; Kim et al., 2015; Paolicelli et al., 2011; Zhan et al., 2014), morphological and density changes induced by Cx3cr1 absence correlate with changes in neural circuit function.”

“67–70,72 Although most studies have focused on the hippocampus, particularly the CA1 and CA3 pyramidal layers and the DG granule Experiments conducted in zebrafish provide additional evidence of microglial activation in the developing brain following seizures.”

“The time course of decline in microglial cell numbers may also vary significantly across regions; most other regions report significant decreases in microglial cell numbers by P21 (Askew et al., 2017; Hagemeyer et al., 2017; Kim et al., 2015; Nikodemova et al., 2015; Riquier & Sollars, 2017), whereas microglial densit…”

“Hippocampal microglia achieve a mature phenotype during the second postnatal week and could be particularly sensitive to activation (Harry, 2013; Kim et al., 2015).”

“…microglia either promote seizures (Abraham, Fox, Condello, Bartolini, & Koh, 2012; Di Nunzio et al., 2021; Eun, Abraham, Mlsna, Kim, & Koh, 2015; Kim et al., 2015) or dampen neuronal hyperactivity (Cserep et al., 2020; Kato et al., 2016; Y. Li, Du, Liu, Wen, & Du, 2012; Merlini et al., 2021)…”

“…and maturation occur prenatally and microglial cells acquire a mature phenotype by the end of prenatal development, whereas, in rats, microglia mature during the first weeks of postnatal development [39–42]. maturation prenatally and microglial cells acquire a mature phenotype by the end of”

“This was also observed in animal models of temporal lobe epilepsy [13].”

“…and experimental studies seem to indicate that the magnitude of microglia reactivity is both, a consequence of as well as an aggravating contributor to the severity of the epileptiform activity and neuronal damage (Brackhan et al., 2016; Johnson et al., 2016; Kim et al., 2015; Lai and Todd, 2008).”

“Microglia in the hip-pocampusarenearlyabsentatbirth,risebyPND7,peakatPND14, and then stabilize at about half the peak level in early adulthood (Kim et al. 2015).”

“Previous studies of epilepsy have reported positive correlations between microglia number and seizure susceptibility during development, as well as seizure mitigation following inhibition of microglial proliferation (Kim et al., 2015; Di Nunzio et al., 2021).”

“53,54 Our results align with these findings and suggest that a loss of inhibitory tone within deeper cortical layers, due to microglial pruning of inhibitory synapses, could contribute to the hyperexcitable state of pyramidal neurons in Layers II and III and promote continued spiking.”

“…microglial activation and limited neuronal injury, whereas seizures during the second postnatal week (P10–P14, coinciding with the peak in postnatal microglial density) strongly provoke neuroinflammatory responses, impair synaptic maturation, and enhance long-term circuit instability [99,100].”

“Furthermore, overactivation of microglia during early development is known to enhance epileptogenicity of neurotoxic insults [35].”

“5, To compare the RNAscope distribution of Il34 and Csf1 (Figure 4) with the expected producing cell, based on their CellChat analysis (Figure 2), the authors show the expression of these two transcripts on the UMAP of the different clusters.”

“Additionally, this developmental window is defined by ongoing synaptic maturation and elimination where microglia are key players in this early synaptic refinement [29–31].”

“23 Nevertheless, while this association was suggested to be causal, evidence provided was at best correlational and lacked direct causal testing.”

“DAMs can negatively affect neurons, potentially leading to the generation or exacerbation of epilepsy (Kim and others 2015).”

“Interestingly, these animals showed increased microglia activation in the hippocampus after seizures https://doi.org/10.3390/cells15090835 compared to the other ages [48].”

“This phenomenon may be related to the dynamic changes in microglial activation during development: in rodents, microglial activation is high during the second postnatal week (d10-d15), followed by a gradual decline in activation (Colonna et al., 2017; Gautier et al., 2012b; Kim et al., 2015).”

“Further, the density of male microglia across the dorsal hippocampus was not changed across conditions at P12 (Figure S1A), even though microglia numbers are likely still expanding at this age (Kim et al., 2015).”

“Therefore, anti-inflammatory therapies targeting the over-production of inflammatory mediators by activated microglia may be an effective age-specific therapeutic strategy to minimize neuronal dysfunction and prevent increased susceptibility to seizures in adult animals (Kim et al. 2015).”

“We first examined synapse engulfment at postnatal day 16–18 (P16–18) as previous studies have found that this age range is when the number of microglia and their phagocytic activity peaks in the hippocampus (Paolicelli et al. 2011; Kim et al. 2015; Weinhard et al. 2018).”

“Interleukin-3 is constitutively expressed by subsets of astrocytes throughout the steady state mouse brain (McAlpine et al. 2021), and yet, microglia proliferate minimally in adult-hood (Ginhoux et al. 2010; Kim et al. 2015; Nikodemova et al. 2015).”

“These two time points were chosen as microglial numbers peak in the hippocampus at this postnatal timepoint (Kim et al., 2015; Weinhard et al., 2018), indicating this is an important time for microglia function in the developing hippocampus.”

“Furthermore, over 394 activation of microglia during early development is known to enhance epileptogenicity of 395 neurotoxic insults(34).”

“Koh and colleagues found that elevated developmental density of microglia in the hippocampus was correlated with increased susceptibility to seizures (Kim et al., 2015), again suggesting that elevated developmental microglial density may be a liability rather than providing protection.”