Hippocampal deficits in neurodevelopmental disorders

Abstract

Neurodevelopmental disorders result from impaired development or maturation of the central nervous system. Both genetic and environmental factors can contribute to the pathogenesis of these disorders; however, the exact causes are frequently complex and unclear. Individuals with neurodevelopmental disorders may have deficits with diverse manifestations, including challenges with sensory function, motor function, learning, memory, executive function, emotion, anxiety, and social ability. Although these functions are mediated by multiple brain regions, many of them are dependent on the hippocampus. Extensive research supports important roles of the mammalian hippocampus in learning and cognition. In addition, with its high levels of activity-dependent synaptic plasticity and lifelong neurogenesis, the hippocampus is sensitive to experience and exposure and susceptible to disease and injury. In this review, we first summarize hippocampal deficits seen in several human neurodevelopmental disorders, and then discuss hippocampal impairment including hippocampus-dependent behavioral deficits found in animal models of these neurodevelopmental disorders.

1. Introduction

Neurodevelopmental disorders (NDs) are a diverse group of disorders resulting from impaired development or maturation of the central nervous system. The causes of NDs are also varied and complex. NDs can result from mutations of single genes, such as in fragile X syndrome (FXS) and Rett syndrome (RTT), or from environmental exposures or injuries, such as fetal alcohol spectrum disorders (FASD) and perinatal ischemia. Most NDs are likely the result of both genetic alterations and environmental impacts^1,2. NDs affect many aspects of biological functions controlled by the brain, including but not limited to challenges with sensory function, motor function, learning, memory, executive function, emotion, anxiety, and social ability. Although these functions are mediated by multiple brain regions, many of them are dependent on proper functioning of the hippocampus, as discussed below.

Extensive research has established critical roles of the mammalian hippocampus in the formation of spatial, temporal, and contextual memories. The hippocampus seems to be important for distinct encoding, retrieval, and consolidation steps during memory formation^3-8. For instance, hippocampal mechanisms can directly influence sensorimotor functions, such as locomotor activity and startle reactivity. Aberrant hippocampal function can lead to neuropsychiatric diseases, in particular psychosis ⁹. In addition, diverse behavioral effects may be associated with different hippocampal subregions. For example, the dorsal hippocampus has a preferential role in certain forms of learning and memory, notably spatial learning, while the ventral hippocampus may have a preferential role in brain processes associated with anxiety-related behaviors ¹⁰. Hippocampal neurons send distinct behavior-contingent information selectively to different target areas. Projections from the ventral hippocampal CA1 to the shell of the nucleus accumbens play a necessary and sufficient role in the formation of social memory¹¹. On the other hand, projections from the ventral hippocampus to the basal amygdala mediate contextual fear behavior, and projections from the ventral hippocampus to the central amygdala mediate retrieval of context-dependent memories of fear¹². Also, the hippocampus integrates with subcortical sites and the prefrontal cortex to play key roles in executive functions. This functional connectivity enables learning-behavior translation, and is in part why hippocampal dysfunctions results in memory and executive deficits¹³.

Long-term potentiation (LTP) is a form of protein synthesis-dependent synaptic plasticity and is considered to be the cellular basis of learning and memory¹⁴. LTP in the hippocampal CA3-CA1 pyramidal neurons is important for learning, and the strength of hippocampal LTP has been used extensively to evaluate the impact of genetic mutations, exposures, diseases, and injuries on brain functions¹⁵

In addition, a unique feature of the mammalian hippocampus is lifelong adult neurogenesis¹⁶. In mammalian experimental models, the subgranular zone of the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles have continuous new neuron production, even though major developmental neurogenesis ceases upon birth in most other brain regions. This postnatal hippocampal neurogenesis, termed adult neurogenesis, has been found in nearly all mammalian species analyzed to date¹⁶, including in humans^17-19. Extensive studies using rodent models have shown that a small number of radial glia-like neural stem cells (NSCs) in the adult hippocampus actively integrate both extrinsic and intrinsic signals to either maintain their quiescent state or divide and give rise to intermediate progenitor cells, which subsequently differentiate into glutamatergic DG granule neurons or astrocytes¹⁶. Although the scale of adult neurogenesis is small, with about 700 new cells generated per day in humans, the lifelong addition of these new cells may have a significant impact on behaviors, neural plasticity, and hippocampal function^18,19. Animal studies using specific genetic ablation or enhancement of adult hippocampal neurogenesis have provided convincing evidence for the importance of adult neurogenesis in hippocampal-dependent learning and working memory^20-22. In addition, a number of recent studies suggest that neurogenesis in dorsal and ventral hippocampus has differential roles in hippocampal-dependent learning and memory²³. These findings are consistent with the notion that sub regions of the adult hippocampus mediate different behaviors, with the dorsal hippocampus important for spatial learning and memory and the ventral hippocampus essential for emotional behavior²⁴.

The hippocampus is known to be vulnerable to hypoxia, stress, toxins, and malnutrition, and it plays a clinically relevant role in children who are born preterm or who have been exposed to alcohol, toxins, or drugs during fetal development^25-27. Also, as discussed in this review, hippocampal dysfunctions are found in NDs resulting from genetic mutations, including FXS, RTT, and Down syndrome (DS)^28-35. The development and progress of noninvasive magnetic resonance imaging (MRI), including volumetric MRI analysis and functional MRI (fMRI), has facilitated the study of hippocampal changes in children with NDs.

Adult neurogenesis is also highly sensitive to genetic mutations and life experiences, providing the cellular basis for gene and environmental interactions that impact behaviors^36,37. Numerous animal studies have shown the important roles of adult hippocampal neurogenesis^38-43. Enhancement of adult neurogenesis via environmental enrichment, physical exercise, nutrition, inhibition of cells death, etc., leads to improved learning and memory, cognitive ability, and reduced anxiety in animal models^44-49. On the other hand, inhibiting adult neurogenesis through genetic mutations, pharmacological inhibition, chronic stress, and disease modeling leads to impaired cognitive functions²¹,⁴⁰,⁵⁰-⁵². Altered adult neurogenesis has been found in a number of animal models of NDs and neuropsychiatric disorders⁵³. However, due to a lack of effective non-invasive methods adult neurogenesis in humans is mostly assessed indirectly through radio carbon dating ¹⁸ and histological analysis of postmortem tissues^17,19. Notably, human adult neurogenesis has yet to be assessed in individuals with NDs.

In this review, we first summarize hippocampal deficits seen in several human NDs, and then discuss the hippocampal changes, particularly hippocampus-dependent behavioral deficits, found in animal models of NDs (Table 1).

Table 1.

Rodent Behavioral Tests for Assessing Hippocampal Related Deficits

Functional Domains	Behavioral Tests	Description	Hippocam pus- dependent	Adult neurogenes is- dependent	Species	Disease models tested	Example References
Associative Learning	Contextual Fear Conditioning	Associate environment with aversive foot shock	Yes	Yes	Mouse Mouse	FXS RTT	(Kazdoba et al., 2014) (Stearns et al., 2007)
	Trace Fear Conditioning	Associate foot shock with context in the presence of time interval between stimuli	Yes	Yes	Mouse	FXS	(Guo et al., 2011; Guo et al., 2012; Zhao et al., 2005)
Spatial Learning and Working Memory	Barnes Maze	Learn and find hiding hole based on spatial cues	Yes	Yes	Mouse	FASD	(Houle et al., 2017)
	Radial Arm Maze	Distinction between closely spaced arms	Yes	Yes	Mouse	FASD	(Berman and Hannigan, 2000)
	Morris Water Maze	Visual-spatial memory task.	Yes	Yes	Mouse Mouse Mouse Mouse+Ra t	FXS ASD RTT FASD	(Kazdoba et al., 2014) (Kwon et al., 2006) (Stearns et al., 2007) (Berman and Hannigan, 2000)
	Novel Location Test	Preference for object in a novel location.	Yes	Yes	Mouse	FXS RTT	(Guo et al., 2011; Guo et al., 2012; Zhao et al., 2005) (Stearns et al., 2007)
Sociability	Three-chamber sociability task	Preference for novel mouse over novel object.	Yes	?	Mouse+Ra t Rat	ASD FXS	(Harony-Nicolas et al.,2017; Jones et al., 2011; Kwon et al., 2006; Sungur et al., 2017; Won et al., 2012) (Tian et al., 2017)
	Caged adult social interaction test	Social preference test for novel mouse or wild-type mouse	?	?	Mouse	ASD	(Tabuchi et al., 2007)
General Cognition	Novel Object Recognition	Preference for novel object.	Yes	?	Mouse Mouse Mouse	ASD FXS RTT	(Sungur et al., 2017) (Kazdoba et al., 2014; Li et al., 2016) (Stearns et al., 2007)
Anxiety	Open field	Overall activity and anxiety	Yes	?	Mouse	FXS	(Guo et al., 2011; Kazdoba et al., 2014)
	Dark/Light Box	Anxiety	Yes	?	Mouse	FXS	(Kazdoba et al., 2014)
	Elevated plus maze	Anxiety	Yes	?	Mouse	FXS	(Kazdoba et al., 2014)

Note: RTT, Rett Syndrome; FXS, fragile X syndrome; FSAD, fetal alcohol spectrum disorders, ASD, autism spectrum disorders.

?, Insufficient information

2. Hippocampal phenotypes in human neurodevelopmental disorders

2.1. Autism and Autism Spectrum Disorder

Autism is a complex developmental disability that appears during the first few years of life⁵⁴. Autism and its related disorders, collectively called autism spectrum disorder (ASD), affect 1 in 68 children in the United States (https://www.cdc.gov/mmwr/volumes/65/ss/ss6503a1.htm), with four times more boys than girls affected⁵⁵. Although about 1000 genes are linked to ASDs, the exact causes are not fully understood. The hypothesis is that a combination of genetic and environmental factors are involved in the pathogenesis and severity of ASDs⁵⁶. Non-invasive MRI has been a key technology for examining abnormalities in the brains of ASD patients, especially children²⁹. A comprehensive literature review of early structural MRI studies shows increased total brain, parieto-temporal lobe, and cerebellar hemisphere volume in juvenile and adult males with ASDs, after adjusting for height, IQ, and intra-cranial volume⁵⁷. Notably, however, only some studies observed a reduction in the volume of the hippocampus of individuals with an ASD⁵⁸. The differences among these results could be due to the different ages of participants included and the relatively low sensitivity of methods used to measure the hippocampus. A more recent study revealed that the relative volume, but not the absolute volume, of the hippocampus is reduced in individuals between the ages of four and 18 who have an ASD, compared to matched controls⁵⁹. Another study of mature adult men (40-64 years old) with ASD found a bilateral reduction in hippocampal volume ⁶⁰. In addition to volume measurements, improved and innovative methods are being developed to study ASD patients. For example, Chaddad et al. used a novel MRI multi-scale image texture analysis to quantify the spatial heterogeneity of brain tissues, including hippocampal volume. They characterized the link between neuroanatomical regions and ASDs and found that five out of 31 prominent subcortical brain regions exhibit significant changes, including the right hippocampus, which is believed to be responsible for encoding spatial relationships⁶¹. In a parallel study, Chaddad et al. performed more detailed image texture analysis on the hippocampus and the amygdala and found that hippocampi are significantly different between those with an ASD and controls with regard to several texture features analyzed⁶². These findings indicate that hippocampal texture features could be used as biomarkers to diagnose and characterize ASD.

2.2. Fragile X syndrome (FXS)

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and the most common single-gene mutation associated with autism⁶³,⁶⁴. FXS results from CGG expansion in the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene on the X chromosome, which leads to epigenetic silencing of the gene⁶³,⁶⁴. Since the identification of FMR1 as the causal gene for FXS in 1991⁶⁵, extensive research, based mainly on animal models, has explored the functions of fragile x mental retardation protein (FMRP) the protein encoded by FMR1 gene^66-69. FMRP is an RNA-binding protein that regulates the translation of a number of mRNAs whose protein products are important for synaptic development, maintenance, and plasticity^70,71. However, despite several research advances, how FMRP deficiency affects human development and the pathogenesis of FXS remains unclear, and there is currently no cure for this ND.

FXS patients exhibit extensive behavioral impairment in executive function, learning, short-term and working memory, and social ability, as well as heightened anxiety⁷². Many of these deficits are related to hippocampal functions and dysfunctions⁷³. Despite advances in our understanding of the molecular changes in FXS, there is limited neuropathological information on FXS patients. To address this issue, Greco et al. analyzed the hippocampus and cerebellum in three men with FXS using histologic, immunochemical, and molecular techniques⁷⁴. They found that individuals with FXS had dysmorphic, enlarged hippocampi, decreased cerebellar size, and preferential atrophy of vermal lobules VI to VII⁷⁴. Another study, however, has found no change in the size of the hippocampus in human FXS⁷⁵. The discrepancies among these studies may result from different methods used and different ages of the patients, similar to those seen in ASD studies. On the other hand, fMRI and behavioral assessments have revealed that individuals with FXS exhibit reduced hippocampal activation during visual memory encoding ⁷⁶ and deficits in episodic memory ⁷⁷. Both of these functions are dependent on the hippocampus. Therefore, the neurological and neuropsychiatric symptoms seen in FXS may, at least in part, be due to abnormal development and dysfunction of the hippocampus.

2.3. Rett syndrome (RTT)

Epigenetic mechanisms, including DNA methylation and histone modification, are known to play significant roles in brain development and function. They also mediate environmental impact on brain functions⁷⁸-⁸⁰. DNA methylation, mostly on CpG dinucleotides (mCG), helps regulate both neurodevelopment and adult neuroplasticity⁸¹-⁸³. Methylated cytosines (mCs) are recognized by proteins with methyl-CpG-binding domains, including methyl-CpG binding protein 2(MeCP2)⁸⁴,⁸⁵. Spontaneous mutations in MeCP2 genes on the X-chromosome are found in 95% of classic RTT patients^86,87. RTT is a progressive neurodevelopmental disorder that almost exclusively affects girls with an incidence of ~1/10,000 live female births⁸⁸. RTT is characterized by an apparently normal development for the first 6–18 months of life followed by a gradual appearance of mental, neurological and physical symptoms⁸⁹. RTT patients exhibit severe mental impairment, slowed growth, motor dysfunction and seizures⁹⁰. Other symptoms include cardiac abnormalities and pronounced breathing disturbance⁹¹. MRI analysis has shown significantly reduced volume in both grey and white matters, including the hippocampus, in RTT, and patients with more severe symptoms exhibit more significant reductions⁹²,⁹³. Magnetic resonance spectroscopy (MRS) has found significant changes in metabolites, including N-acetyl aspartate (NAA), in the hippocampus of RTT patients⁹⁴. Recently, a MeCP2-mutant monkey model has been created through genome editing. MRI analysis has shown that the size of the hippocampus, particularly the posterior parahippocampal gyrus, is reduced in these monkeys⁹⁵, similar to what has been found in human patients. In addition, histological studies of human postmortem tissues have found that pyramidal neurons in the cortex and the hippocampus of RTT individuals have reduce dendritic complexity⁹⁶-⁹⁸. Chapleau et al showed hippocampal CA1 pyramidal neurons from postmortem female patients had reduced dendritic spine density compared to controls⁹⁸. To confirm this result, they introduced RTT-mutant MeCP2 into hippocampal neurons of rat brain slices and found that expression of mutant MeCP2 leads to reduced dendritic spine density compared to wild type MeCP2⁹⁸. Hence, hippocampal deficits are apparent and severe in RTT and likely contribute to neurological and cognitive symptoms.

2.4. Down syndrome (DS)

Down syndrome, or trisomy 21, is the most common human aneuploidy condition leading to intellectual disability ⁹⁹. Chromosome 21 is the smallest human chromosome¹⁰⁰, which may explain why trisomy of this chromosome is compatible with life, and results in only mild-to moderate intellectual disability. DS patients exhibit cognitive impairment, motor dysfunction, increased seizure risk, and increased incidence of Alzheimer’s disease^101,102. There have been a large number of MRI analyses for DS, and these studies have shown that DS patients have reduced volume of the hippocampus¹⁰³. In addition, DS patients with dementia exhibit more severe reductions of hippocampus volume compared to DS patients without dementia¹⁰³-¹⁰⁵.

2.5. 15q syndromes

The human chromosome 15q11-q13 region is prone to copy number variations, which can be either deletions or duplications, that lead to several distinct neurodevelopmental disorders with some overlapping phenotypes¹⁰⁶. In addition, part of this genomic region is imprinted and its haploinsufficiency leads to either Angelman syndrome (AS), if it is a maternal deletion, or Prader-Willi syndrome (PWS), if it is a paternal deletion¹⁰⁷. Genetic changes in this 15q region lead to several characteristic NDs, and all of them exhibit hippocampal deficits to some extent, as discussed below.

Angelman syndrome, which affects 1 in 12,000-20,000 people, results mostly from maternal deficiency of UBE3A, a gene located on 15q11-q13¹⁰⁸. Children with AS seem to develop normally during the first few months of life with clinical symptoms manifesting later in development¹⁰⁹. Children with AS have delayed development, impaired speech, defective movement, and unique behavioral features including a happy demeanor and frequent laughing¹¹⁰. Microcephaly is a common feature of children with AS¹¹¹. Therefore, the overall brain volume, including global white matter and gray matter (including the hippocampus), in children with AS is smaller compared to normal children¹¹².

Prader-Willi syndrome (PWS) has a prevalence of around 1 in 15,000 births and affects males and females equally. It can result from either paternal genetic deletion of 15q11-q13 or maternal uniparental disomy or imprinting defects¹¹³. PWS patients exhibit profound early childhood-onset obesity caused by excessive and pathological overeating. They also show intellectual disability, including speech delay, learning disabilities (low or very low intelligence and deficits in adaptive behaviors without reference to etiology), and cognitive deficiencies (depressed general cognitive functioning or IQ, processing deficits, including problems with short-term memory, language processing deficits, and difficulty with higher order processing)¹¹⁴. Structural MRI studies have shown that PWS patients have reduced gray matter volume that encompasses several major brain regions, including the hippocampus¹¹⁵. Functional MRI studies found that PWS patients have reduced neural network connectivity in several cortical regions, including the prefrontal cortex and the hippocampus¹¹⁶. PWS is one of a very few well defined genetic causes of obesity. Therefore, food-induced brain changes have been studied extensively¹¹⁷. Studies have shown that several brain regions, including the medial prefrontal cortex, amygdala, and hippocampus are activated by food or either sight or smell of food, but there was a decreased response to food stimulus after eating^118-120. It has been shown that PWS patients exhibit abnormal food stimuli-induced activity in these brain regions compared to typical individuals with normal body weight¹²¹. Another study comparing PWS versus non-PWS obese individuals discovered that PWS patients show hyperactivation in subcortical reward circuitry but hypoactivation in cortical regions including the hippocampus after eating¹²².

Patients with maternal copy number gains of 15q11-q13 are diagnosed with 15q duplication syndromes (Dup15q) and exhibit global developmental delay, intellectual disability, autistic phenotypes, and epilepsy¹⁰⁶. The prevalence of Dup15q is estimated to be 1 in 30,000 with a sex ratio of 1:1¹²³. MRI studies showed that Dup15q patients have increased hippocampus sclerosis, a lesion possibly caused by excitotoxicity¹²⁴.

2.6. Fetal alcohol spectrum disorders (FASDs)

Fetal alcohol spectrum disorders (FASDs) is the umbrella term covering the full range of deficits resulting from prenatal exposure to alcohol¹²⁵. The most severe form of FASD is fetal alcohol syndrome (FAS), which affects 0.5-2 per 1000 births in the US but varies across the world, with the highest reported rates in South Africa (60 per 1000 births)^126,127. Individuals with FASDs exhibit abnormal appearance, short height, low body weight, small head size, poor coordination, low intelligence, behavior problems, and problems with hearing or seeing¹²⁸. Surveys from the United States have found that about 19.6% of women drink alcohol while pregnant¹²⁹. The drinkers reported significantly higher levels of depressed mood and more problems with alcohol than their counterparts who abstained¹²⁹. Studies have shown that even low to moderate levels of exposure to alcohol can have detrimental effects¹³⁰. The incidence of FASD is unclear because of the difficulty in identifying the affected individuals, but the prevalence is likely high. Individuals with FASD have a wide range of deficits^130,131, including reduced IQ, cognitive impairments, and social and behavioral difficulties as well as predisposition to mental health issues^130,131. Individuals with FASD typically do not complete their education and are often unemployed and many of them experience depression, increased suicide risk, and trouble with the legal system. Consequently, FASD imposes an enormous life-long burden on families and society^132,133.

Neuroimaging analysis has identified significant abnormality in brain regions of individuals with FASD with overall reduction in brain volumes¹³⁴. The deficits associated with FASD point to impairment of hippocampal functions¹³⁵. MRI assays have shown reduced hippocampal volumes in individuals with FASD, which correlated with learning and memory deficits. Magnetic resonance spectroscopy (MRS) has also identified changes in metabolites in the hippocampus of FASD individuals¹³⁴. A recent MRI analysis has confirmed that the sizes of all brain structures analyzed were reduced in individuals with FASD compared to controls, including gray and white matter of the cerebrum and cerebellum, and all deep gray matter, including the hippocampus, amygdala, thalamus, caudate, putamen, and pallidum¹³⁶. Although alcohol exerts extensive effects on developing brains, these research findings indicate that its impact on the hippocampus likely contributes to the learning and memory deficits seen in FASD.

3. Hippocampal phenotypes in animal models for neurodevelopmental disorders

3.1. ASD

Alterations in neural network connectivity and memory function are frequently observed in autism patients and often involving the hippocampus¹³⁷. However, the changes that occur during early brain development that lead to disrupted brain functions in ASD remain largely unclear. A large number of animal models for ASD have been developed and used for investigating the changes in developing brains¹³⁸. Studies have shown that rodents exposed to valproic acid (VPA) during fetal development go on to exhibit many features of autism. Therefore, prenatal exposure to VPA has been used to establish model of autism in rodents to extensively study the underlying mechanisms behind autism. However, since different dosages and timing of prenatal VPA treatment may result in various phenotypes, interpretations of results should be made with caution¹³⁹,¹⁴⁰. Advancement in human genetics has led to the discovery of many genes associated with ASD, and based on these discoveries, over 1000 autism genetic mouse models and over 200 rat genetic models have been created. Simons Foundation Autism Research Initiative (SFARI) has identified 65 high–ranking candidate risk genes for ASD, with SHANK3 and PTEN among the high confidence genes (https://www.sfari.org/resource/sfari-gene/).

SHANK proteins are scaffolding proteins, located at excitatory synapses, and are crucial for proper synaptic development and function. The SHANK proteins SHANK1, 2, and 3 are encoded by separate genes in both human and mouse genomes¹⁴¹. Mutations in any of the three SHANK genes have been associated with autism¹⁴². Mutant mouse models have been generated to study the functions of these genes, and these mutant mice shared apparent hippocampal deficits. Hippocampal neurons of Shank1null mice exhibited altered postsynaptic density protein (PSD) composition and thinner PSDs, reduced size of dendritic spines, and decreased AMPAR-mediated excitatory synaptic transmissions¹⁴³. In addition, Shank1null mice exhibit decreased reduced tonic firing rates of parvalbumin (PV) positive neurons in the hippocampus¹⁴⁴. At the behavioral level, Shank1 null mice are defective in hippocampus-dependent contextual fear memory and object recognition memory¹⁴⁵,¹⁴⁶. SHANK1 protein is highly co-localized in PV-expressing fast-spiking inhibitory interneurons in the hippocampus. Lack of SHANK1 in these cells leads to a decrease in excitatory synaptic inputs and inhibitory synaptic outputs to pyramidal neurons at the functional level, together with molecular changes, including the downregulation of the postsynaptic proteins GKAP, PSD-95, and gephyrin¹⁴⁴. Shank2 (also known as ProSAP1) null mice are extremely hyperactive and display profound autistic-like behavioral alterations including repetitive grooming as well as abnormalities in vocal and social behaviors¹⁴⁷. At the cellular level, neurons in the hippocampus of Shank2 null mice exhibit reduced spine density and impaired NMDAR-dependent synaptic plasticity ¹⁴⁷,¹⁴⁸. Because SHANK3 has the strongest link to ASD, several mouse lines with distinct Shank3 gene mutations have been developed to help understand ASD-related neurobiology. These mouse models showed ASD-related behavioral phenotypes, including impaired social behavior, increased repetitive behaviors, motor deficits, as well as altered synaptic transmission and neuronal morphology in the hippocampus¹⁴². To better study ASD-related behaviors, Shank3-deficient rats have been created. These rats exhibit impaired long-term social recognition memory and attention, and reduced synaptic plasticity in the hippocampal-medial prefrontal cortex pathway¹⁴⁹. The peptide hormone oxytocin has been shown to improve social memory, enhance social reward, and modulate social attention in human and non-human primates¹⁵⁰-^152,153, Harony-Nicolas et al utilized oxytocin to rescue social and attention deficits in Shank3 deficient rats¹⁴⁹. Several studies have identified the downstream pathways and proteins associated with the SHANK3 network. It has been shown that Shank3 deletion impairs the function and synaptic localization of mGluR5 in several brain regions, including the hippocampus in mice¹⁵⁴,¹⁵⁵ and rats¹⁵⁶. Cope et al has also assessed adult hippocampal neurogenesis in Shank3^+/ΔC transgenic mice that have deletion of a Homer-binding region, a mutation also found in some humans with autism and found that both Shank3^+/ΔC mice exhibit significant reduction in the number of radial glia-like neural stem cells and immature neurons in the DG of the ventral hippocampus¹⁵⁷. Therefore, the phenotypes of animal models with SHANK protein mutations or deletions support the involvement of the hippocampus in the pathology of ASD.

The phosphatase and tensin homolog on chromosome ten (PTEN) gene is a tumor suppressor, and its mutation leads to many human cancers¹⁵⁸. PTEN mutations are associated with a number of neurological conditions, including ASD, megalocephaly and epilepsy as well¹⁵⁹. Homozygous deletion of PTEN in mice leads to embryonic lethality, and heterozygote mutant mice are prone to tumor formation¹⁶⁰,¹⁶¹, demonstrating the important role of this protein in development. PTEN catalyzes dephosphorylation of phosphatidylinositol 3, 4, 5 trisphosphate, a critical cellular signaling molecule that controls many cellular pathways¹⁶². Extensive studies using mouse models have shown that PTEN-induced hyperactivation of the mammalian target of rapamycin (mTOR) pathway leads to abnormalities in neuronal proliferation, survival, growth and plasticity in the brain including in the hippocampus¹⁶³. PTEN deletion in hippocampal DG and CA3 neurons leads to dysregulation of synaptic plasticity of DG granule neurons, which seems to precede visible morphological changes¹⁶⁴. Several laboratories have investigated the role of PTEN in adult hippocampal neurogenesis. Conditional deletion of PTEN in Nestin-expressing cells in mice leads to increased proliferation, reduced differentiation and stem cell depletion ¹⁶⁵. In addition, acute knockdown of PTEN in new neurons in the adult hippocampus leads to hyper-excitability¹⁶⁶. Furthermore, although it is not known whether adult hippocampal neurogenesis regulates social interaction, neurogenesis during adolescence has been found to be important in developing social ability¹⁶⁷. Interestingly, deletion of PTEN in new DG neurons born in 4-weeks old mice leads to impaired social interaction¹⁶⁵, and is a core phenotype of autism. Therefore, juvenile neurogenesis may contribute to social impairment seen in ASD.

Neurexin (NRXN) and Neuroligin (NLGN) family proteins cooperatively function at synaptic terminals, and the absence of both is strongly associated with the occurrence of ASDs, especially NRXN1 and NLGN3 ^168-170. Mammals have three NRXN genes (NRXN1, NRXN2 and NRXN3), and all of them produce longer α- and shorter β- form of proteins through transcription from independent promoters¹⁷¹. Several different lines of NRXN knockout (KO) mice have been developed. α-NRXN triple KO mice die shortly after birth due to respiratory failure ¹⁷². β-NRXN triple KO mice are ~80% smaller in body weight, exhibit impairment in hippocampus-dependent contextual fear memory and reduction in excitatory neurotransmitter release in the hippocampus^173,174. NRXN3 KO mice lacking both α- and β- forms show reduced AMPA receptor function in hippocampal neurons due to a deficit of NRXN3-mediated trans-synaptic regulation of postsynaptic AMPA receptors¹⁷⁵.

There are four NLGNs in mammals¹⁷⁶. The NGLN3 mutant (R451C allele) mouse model, carrying a single rare point mutation of NLGN3 was originally discovered in patients with ASDs. This mouse model shows impaired social interaction but enhanced spatial learning abilities with increased LTP and excitatory transmission in the hippocampal region ¹⁷⁷,¹⁷⁸. NLGN3 R451C mice also display impairment in tonic endocannabinoid signaling at inhibitory synapses formed between interneurons and hippocampal pyramidal neurons, and this endocannabinoid signaling may be essential for maintaining excitation and inhibition balance¹⁷⁹. On the other hand, NLGN3 R704C knockin mice, which carry another autism mutation, show a selective reduction of AMPA receptor-mediated synaptic transmission in the hippocampus¹⁸⁰. Mice deficient in other Neuroligins (NGLN 1, 2, 4) also exhibit hippocampal deficits, including synaptic plasticity changes in the CA regions and impaired hippocampal-dependent spatial learning¹⁷².

In addition to the genetic models created based on human ASD gene discoveries, phenotypic screening using inbred strains as part of the Jackson Lab Phenome project has identified BTBR^T+tf/J (BTBR) mice as the strain that shows the strongest ASD-related phenotypes. These mice show impaired social interaction and communication, increased repetitive behaviors and learning and memory deficits^138,181. Additional studies show that BTBR mice also have hippocampal deficits, including enhanced development of dendritic arbor of hippocampal CA1 pyramidal neurons ¹⁸², greater relative hippocampal volume ¹⁸³,¹⁸⁴ and increased local connectivity in the hippocampal commissure¹⁸⁵. BTBR mice also show reduced GABAergic neurotransmission in the hippocampus. Low-dose benzodiazepine treatment, which increases inhibitory neurotransmission through activation of GABAA receptors, improves deficits in social interaction, repetitive behavior, and spatial learning¹⁸⁶. Therefore, mouse models of ASD exhibit altered hippocampal functions and hippocampal associated behavioral deficits.

3.2. Fragile X syndrome (FXS)

Animal models for FXS have been developed in Drosophila, zebrafish, mice, and rats. Most FXS behavioral modeling has been done using mouse models with a large number of publications ^187-190. In the most widely used mouse model (Fmr1 KO), exon 5 was interrupted by the positive selection marker gene neomycin (neo), resulting in a mutant Fmr1 mRNA, which fails to produce FMRP protein¹⁹¹. The Fmr1 KO mouse model has been extensively tested and compared with human FXS phenotypes. These mice exhibit several human FXS-related deficits but not others¹⁹². Several mouse behavioral tests have been used to assess hippocampus-dependent cognitive functions in Fmr1 KO mice. Although some studies have shown that Fmr1 KO mice are impaired in hippocampus-dependent contextual fear conditioning, delayed trace conditioning, Morris Watermaze, novel object recognition, and novel location tests, other laboratories have found no change or even opposite results (Figure 1, Table 1) (Please refer to Kazdoba et al for a comprehensive review on variabilities in behavioral deficits of Fmr1 KO mice¹⁹²).

Figure 1.

Comparison of behavioral deficits, particularly hippocampus-dependent deficits, observed in human fragile X syndrome patients and those found in Fmr1 mutant mouse models.

Hippocampus-dependent behaviors are indicated with asterisks (*). Adult hippocampal neurogenesis-dependent behaviors are indicated with an arrow (→).

Both the hippocampus and isolated hippocampal neurons of rodents have been widely used to study FXS. Fmr1 KO mice show enhanced protein synthesis–dependent long-term depression of synaptic transmission in the hippocampus, which results from the alteration of mGluR signaling¹⁹³-¹⁹⁵. A consistent discovery is that the absence of FMRP leads to exaggerated mGluR5 signaling, enhanced protein synthesis, and changes in synaptic plasticity. Based on the mGluR5 theory¹⁹⁶, a number of pharmacological preclinical studies have been done and show remarkable effects in correcting Fmr1 KO mice phenotypes through mGluR5 inhibition¹⁹⁷-¹⁹⁹. Subsequently, large clinical trials were carried out between 2008 and 2014, which aimed at blocking mGluR5²⁰⁰ using molecules such as two mGluR5 antagonists, basimglurant²⁰¹ and mavoglurant²⁰². Both hippocampal neurons isolated from neonatal Fmr1 KO mice and neurons in the hippocampus of Fmr1 KO mice exhibit the immature dendritic spine phenotypes shown by limited studies of human postmortem tissues ²⁰³-²⁰⁵. Meredith et al investigated whether abnormal spine morphology observed in the Fmr1 KO mouse is paralleled by changes in basic functional synaptic properties at three different developmental stages of hippocampal development. They observed that inhibition of mGluR5 rescued synaptic plasticity when treatment was done at 2 weeks but not at 1 week or 8-10 weeks of age, pointing to the importance of treatment timing for certain FXS phenotypes²⁰⁶.

Several laboratories, including our own, have investigated the role of FMRP in adult hippocampal neurogenesis and found that FMRP deficiency leads to changes in adult neurogenesis²⁰⁷-²⁰⁹. FMRP deficiency in adult hippocampal NSCs led to increased proliferation of adult NSC and altered NSC specification from neurons to astrocytes^52,208. In addition, selective deletion of FMRP from adult NSCs and new neurons leads to impaired performance of trace conditioning, radial arm maze, novel object recognition, and novel location test which are tasks dependent on an intact hippocampus^52,189. On the other hand, restoration of FMRP only in adult new neurons partially rescued some of these deficits, supporting an important role of FMRP in adult neurogenesis and cognition⁵². Using NSCs isolated from adult hippocampus, Guo et al showed that FMRP deficiency in adult NSCs leads to impaired Wnt signaling and inhibition of GSK-3-β, an inhibitor of Wnt signaling, can rescue both adult neurogenesis and hippocampus-dependent learning²⁰⁸,²¹⁰. Recently, Li et al discovered that FMRP controls the activation of adult NSCs which is important for rebalancing NSC quiescence and activation¹⁸⁹. We showed that FMRP maintains the NSC pool through regulation of the MDM2-P53 pathway. A small molecule, Nutlin-3, rescued both neurogenic and cognitive deficits in fragile X syndrome mice¹⁸⁹. Subsequently, we have found that loss of FMRP depletes adult hippocampal NSCs and leads to cognitive impairment, both of which are associated with dysregulation of EP300, a histone acetyltranferase, and targeting of HDAC1, a histone deacetylase, by MDM2, leading to elevated histone H3 and H4 acetylation. Rebalancing histone acetylation with the small molecules Nutlin-3 or Curcumin inhibits MDM2 and EP300 and rescues both neurogenic and cognitive deficits in Fmr1 mutant mice²¹¹. Fmr1 KO rats have been generated using CRISPR/Cas9 technology and these rats have impaired long-term synaptic plasticity and hippocampus-dependent learning²¹². Therefore, hippocampal dysfunctions likely underscore the extensive cognitive deficits in FXS.

3.3. Rett syndrome (RTT)

Methyl-CpG binding protein 2 (MECP2) binds to methylated DNA in the genome leading to transcriptional changes. Therefore, it is an important reader and interpreter of DNA methylation across the genome²¹³. Several mouse models of RTT have been created to resemble human conditions, Interestingly, complete MeCP2 deficiency (null mutant), partial MeCP2 deletions, point mutations, phosphorylation site mutations, or haploinsufficiency recapitulate many neurological and behavioral deficits of human RTT²¹⁴-²¹⁸. In addition, conditional mutant mouse models have been generated and used to demonstrate the function of MeCP2 in specific cell types at different developmental stages, as well as identify MeCP2 regulated targets in various brain regions and cell types²¹⁹-²²³. Furthermore, these mouse studies show that MeCP2 mutations or deficiency in hippocampal neurons leads to extensive functional deficits, including impaired dendritic and spine development, altered excitation inhibitor balance and changed synaptic plasticity²²⁴-²²⁷. In addition, mice with MeCP2 deletion specifically in GABAergic neurons has impaired hippocampal LTP and exhibit repetitive behaviors and impaired hippocampal learning and memory²²⁸.

One of the most consistent behavioral phenotypes of MeCP2-deficient mice is impaired contextual fear conditioning, which is dependent on both the hippocampus and adult hippocampal neurogenesis²²⁹. Several studies have investigated the role of overall MeCP2 expression in regulating hippocampal neurogenesis²³⁰,²³¹. Although MeCP2 null mice did not show impaired NSC proliferation or differentiation, new adult hippocampal neurons exhibit reduced dendritic complexity and spine density²³². MeCP2 regulates maturation of adult born neurons by repressing several microRNAs²³¹,²³³. In post-mitotic neurons, depolarization-induced Ca²⁺ influx through voltage-gated calcium channels (VGCCs) has been shown to trigger phosphorylation of MECP2 at serine 421 (S421)²³⁴,²³⁵, which is required for regulating synaptogenesis, dendritic morphology, synaptic scaling, long-term potentiation and spatial memory in the adult mouse brain²³⁶-²³⁸. However, it is unclear whether phosphorylation at any of the identified sites on MECP2 can be induced by signals other than neuronal activity in other cell types, and what functions MECP2 phosphorylation may have in those contexts. Li et al analyzed adult neurogenesis in mice with S421 mutation of MECP2 and found that S421 phosphorylation regulates the proliferation and neural differentiation of adult NPCs by controlling key factors of Notch signaling pathways²³⁹. This finding suggests that MECP2 has cell type-specific roles in adult neurogenesis. Deep brain stimulation of the hippocampus in symptomatic RTT mice restored hippocampal synaptic plasticity, and adult hippocampal neurogenesis, and rescued hippocampus-dependent contextual fear conditioning.²⁴⁰. Although it remains to be determined whether this treatment effect is associated with adult neurogenesis, these exciting results underscore the significance of hippocampal deficits and rescue in RTT.

3.4. Down syndrome

Several Down syndrome (DS) mouse models have been developed: Ts65Dn²⁴¹ and Ts1Cj²⁴² mice carry larger trisomic segments of mouse chromosome 16, which contains most regions orthologous to human chromosome 21. Ts2Cje²⁴³ mice carry a smaller segment without APP. Ts65Dn mice exhibit extensive cognitive deficits similar to humans with DS and therefore have been used extensively as a DS model²⁴⁴. Quantitative MRI studies have been used to show that Ts65Dn mice exhibit impaired cholinergic circuits, including those in the hippocampus²⁴⁵. Diffusional kurtosis imaging (DKI) analysis shows that Ts65Dn mice have changes in the hippocampus at all ages analyzed (2 months to 8 months)²⁴⁶. Ts65Dn mice exhibit excessive inhibition in hippocampal circuits and show severe impairment of LTP, which can be normalized by inhibition of GABAA receptors²⁴⁷. A study using four mouse models carrying different regions of human chromosome 21 showed that none of these strains exhibited all impairments seen in human individuals with DS, but some strains show reference memory, working memory, and episodic memory changes. Nevertheless, the volume of hippocampus and cerebellum are reduced in all strains²⁴⁸. Interestingly, Ts65Dn, Ts1Cje, and Ts2cje mice all exhibited reduced adult hippocampal neurogenesis²⁴⁹-²⁵². Since either treatment with fluoxetine or physical exercise, known to enhance adult neurogenesis, can rescue learning memory deficits of Ts65Dn mice²⁵⁰-²⁵², promoting adult hippocampal neurogenesis might be a potential therapeutic method for DS.

3.5. 15q syndromes

Mice with maternal deletion of UBE3A have been used to study Angelman syndrome (AS)²⁵³. These mice show increased oxidative stress in the hippocampus, as measured by MRI²⁵⁴. Impaired adult neurogenesis has also been found in an AS mouse model; this phenotype can be rescued with fluoxetine^255,256. Conditional reinstatement of UBE3A showed that restoring UBE3A in various ages resulted in various levels of behavioral rescue²⁵⁷. To rescue anxiety, repetitive behavior and epilepsy phenotypes, UBE3A had to be reinstated early in development but hippocampal synaptic plasticity could be restored at any age, including adult (>8 weeks old)²⁵⁷. However, hippocampal-dependent neurogenesis or learning was not assessed in these mice. It will be interesting to determine whether the plasticity of the hippocampus is due to the presence of adult neurogenesis.

3.6. Fetal alcohol spectrum disorder (FASD)

Animal models for FASD have been created to study the pathogenesis, mechanism, and treatment involving neurodevelopmental abnormalities, neurobehavioral problems, learning difficulties, cognitive problems, intellectual disability, and sleep disorders and associated behavioral impairments²⁵⁸. Many neural and cognitive deficits of FASD are related to disruptions of hippocampal functions. Therefore, extensive research has been focused on the impact of FASD on the hippocampus²⁵⁹,²⁶⁰.

A large number of studies have assessed adult hippocampal neurogenesis in rodent models of FASD²⁶¹. Fetal alcohol exposure appears to cause a reduction in the volume of the hippocampus, which is thought to result from either an overall reduction in cell proliferation or an increase in cell death, leading to impairments in memory functions²⁶²,²⁶³. Chronic prenatal alcohol exposure reduces Doublecortin (DCX)-positive cells, suggesting the rate of conversion of proliferative cells to immature neurons in the hippocampus is impaired²⁶⁴. Kajimoto et al utilized inducible nestin-CreER^T2 transgenic mice to determine the stage-specific impact of prenatal alcohol exposure on the stepwise maturation of adult hippocampal progenitors²⁶⁵. Using a limited alcohol access “drinking-in-the-dark” model of FASD, the study confirmed previous findings that moderate prenatal alcohol exposure had no effect on adult neurogenesis under standard housing conditions, but it did abolish the neurogenic response to enriched environments (EE)²⁶⁵. The effect of alcohol on immune activation in the developing CNS has just begun to be investigated. Drew et al demonstrated for the first time that alcohol induces neuroinflammation with elevated levels of pro-inflammatory molecules and microglial activation in many brain regions²⁶⁶. Neuroinflammation is well known to inhibit adult hippocampal neurogenesis and hippocampus-dependent cognitive functions¹⁶. This study has revealed that the PPAR-c agonist pioglitazone suppresses alcohol-induced neuroinflammation, suggesting that pioglitazone, and perhaps other anti-inflammatory therapeutics, may be effective in the treatment of FASD²⁶⁶. Therefore, hippocampal deficits might be a therapeutic target for FASD.

4. Summary and perspectives

In this review, we hope to convey that hippocampal deficits are prevalent in both human patients with NDs and animal models of NDs. A comparison between human data and animal models allows us to investigate the causes and consequences of changes in hippocampal structures and functions and establish direct causal links between these hippocampal changes with hippocampus-dependent behavioral deficits. Several key questions remain to be addressed. Do hippocampal changes arise from the NDs or precede and predispose toward NDs? What cellular mechanisms underlie the overall increase or decrease in hippocampal volume? In addition, several considerations emerge from this review. How does the study of certain NDs with clear genetic causes, for example, RTT, FXS, and Angelman syndrome, inform our understanding of complex disorders with unclear genetic causes, such as FASD and autism? How can the study of NDs lead to breakthroughs in our understanding of the basic biological mechanisms regulating human development and in the prevention of NDs? Although the extensive use of animal models facilitates our understanding of human NDs, animal models are limited in how much they can reveal about some of the most fundamental aspects of development, genetics, pathology, and disease mechanisms that are unique to humans.

GABAergic inhibition shapes cortical-hippocampal neural activity, which is highly associated with important aspects of neural information processing²⁶⁷. GABA dysfunction has been implicated in many types of NDs, such as ASD¹⁸⁶, DS²⁴⁷, RTT²²⁸ and FXS²⁶⁸. Disruption of hippocampal GABA function by intra-hippocampal infusion of subconvulsive doses of a GABA-A antagonist has recently been shown to disrupt attentional function and hippocampus-dependent place memory performance in rats²⁶⁹. The development of human pluripotent stem cells (hPSCs) and gene editing methods has greatly advanced our ability to study human diseases; however, whether these cell culture-based models are sufficiently complex to correctly mimic human brain development is unclear. Thus, how to best model NDs remains controversial. Since most NDs likely result from a combination of genetic, biological, psychosocial, and environmental risk factors, how do we evaluate the impact of these risk factors and develop treatments for NDs long past the developmental period? Despite the extensive hippocampal deficits observed in human NDs and the widespread observations of deficits in adult hippocampal neurogenesis in animal ND models, neurogenic changes in human NDs remain unexplored, and hippocampal neurogenesis-focused therapeutic interventions have yet to be developed. Much of our knowledge about hippocampal deficits is associative and superficial. We hope that with rapid technological advances in genomics, brain imaging, circuit-specific and cell type-specific manipulation techniques, and more refined and relevant behavioral analysis methods for both animal models and human patients, we can systematically tackle these questions soon.

Highlights.

The mammalian hippocampus has an important role in learning and cognition.

Hippocampal deficits are found in several human neurodevelopmental disorders,

Animal models for neurodevelopmental disorders exhibit hippocampal deficits.

Targeting the hippocampus helps to understand etiology and development treatment