Hippocampal area CA2: interneuron disfunction during pathological states.

Hippocampal area CA2 plays a critical role in social recognition memory and has unique cellular and molecular properties that distinguish it from areas CA1 and CA3. In addition to having a particularly high density of interneurons, the inhibitory transmission in this region displays two distinct forms of long-term synaptic plasticity. Early studies on human hippocampal tissue have reported unique alteration in area CA2 with several pathologies and psychiatric disorders. In this review, we present recent studies revealing changes in inhibitory transmission and plasticity of area CA2 in mouse models of multiple sclerosis, autism spectrum disorder, Alzheimer's disease, schizophrenia and the 22q11.2 deletion syndrome and propose how these changes could underly deficits in social cognition observed during these pathologies.

Environmental enrichment and social isolation modulate inhibitory transmission and plasticity in hippocampal area CA2.

Environmental factors are well-accepted to play a complex and interdependent role with genetic factors in learning and memory. The goal of this study was to examine how environmental conditions altered synaptic plasticity in hippocampal area CA2. To do this, we housed adult mice for 3 weeks in an enriched environment (EE) consisting of a larger cage with running wheel, and regularly changed toys, tunnels and treats. We then performed whole-cell or extracellular field recordings in hippocampal area CA2 and compared the synaptic plasticity from EE-housed mice with slices from littermate controls housed in standard environment (SE). We found that the inhibitory transmission recruited by CA3 input stimulation in CA2 was significantly less plastic in EE conditions as compared to SE following an electrical tetanus. We demonstrate that delta-opioid receptor (DOR) mediated plasticity is reduced in EE conditions by direct application of DOR agonist. We show that in EE conditions the overall levels of GABA transmission is reduced in CA2 cells by analyzing inhibition of ErbB4 receptor, spontaneous inhibitory currents and paired-pulse ratio. Furthermore, we report that the effect of EE of synaptic plasticity can be rapidly reversed by social isolation. These results demonstrate how the neurons in hippocampal area CA2 are sensitive to environment and may lead to promising therapeutic targets.

A hypothalamic novelty signal modulates hippocampal memory.

The ability to recognize information that is incongruous with previous experience is critical for survival. Novelty signals have therefore evolved in the mammalian brain to enhance attention, perception and memory1,2. Although the importance of regions such as the ventral tegmental area3,4 and locus coeruleus5 in broadly signalling novelty is well-established, these diffuse monoaminergic transmitters have yet to be shown to convey specific information on the type of stimuli that drive them. Whether distinct types of novelty, such as contextual and social novelty, are differently processed and routed in the brain is unknown. Here we identify the supramammillary nucleus (SuM) as a novelty hub in the hypothalamus6. The SuM region is unique in that it not only responds broadly to novel stimuli, but also segregates and selectively routes different types of information to discrete cortical targets-the dentate gyrus and CA2 fields of the hippocampus-for the modulation of mnemonic processing. Using a new transgenic mouse line, SuM-Cre, we found that SuM neurons that project to the dentate gyrus are activated by contextual novelty, whereas the SuM-CA2 circuit is preferentially activated by novel social encounters. Circuit-based manipulation showed that divergent novelty channelling in these projections modifies hippocampal contextual or social memory. This content-specific routing of novelty signals represents a previously unknown mechanism that enables the hypothalamus to flexibly modulate select components of cognition.

The mechanisms shaping CA2 pyramidal neuron action potential bursting induced by muscarinic acetylcholine receptor activation.

Recent studies have revealed that hippocampal area CA2 plays an important role in hippocampal network function. Disruption of this region has been implicated in neuropsychiatric disorders. It is well appreciated that cholinergic input to the hippocampus plays an important role in learning and memory. While the effect of elevated cholinergic tone has been well studied in areas CA1 and CA3, it remains unclear how changes in cholinergic tone impact synaptic transmission and the intrinsic properties of neurons in area CA2. In this study, we applied the cholinergic agonist carbachol and performed on-cell, whole-cell, and extracellular recordings in area CA2. We observed that under conditions of high cholinergic tone, CA2 pyramidal neurons depolarized and rhythmically fired bursts of action potentials. This depolarization depended on the activation of M1 and M3 cholinergic receptors. Furthermore, we examined how the intrinsic properties and action-potential firing were altered in CA2 pyramidal neurons treated with 10 µM carbachol. While this intrinsic burst firing persisted in the absence of synaptic transmission, bursts were shaped by synaptic inputs in the intact network. We found that both excitatory and inhibitory synaptic transmission were reduced upon carbachol treatment. Finally, we examined the contribution of different channels to the cholinergic-induced changes in neuronal properties. We found that a conductance from Kv7 channels partially contributed to carbachol-induced changes in resting membrane potential and membrane resistance. We also found that D-type potassium currents contributed to controlling several properties of the bursts, including firing rate and burst kinetics. Furthermore, we determined that T-type calcium channels and small conductance calcium-activated potassium channels play a role in regulating bursting activity.

Routing Hippocampal Information Flow through Parvalbumin Interneuron Plasticity in Area CA2.

The hippocampus is critical for the formation of episodic memory. It is, therefore, important to understand intra-hippocampal circuitry, especially in the often overlooked area CA2. Using specific transgenic mouse lines combined with opto- and chemogenetics, we show that local plasticity of parvalbumin-expressing interneurons in area CA2 allows CA3 input to recruit CA2 pyramidal neurons (PNs), thereby increasing the excitatory drive between CA3 and CA1. CA2 PNs provide both stronger excitation and larger feed-forward inhibition onto deep, compared with superficial, CA1 PNs. This feed-forward inhibition, largely mediated by parvalbumin-expressing interneurons, normalizes the excitatory drive onto deep and superficial CA1 PNs. Finally, we identify a target of CA2 in area CA1, i.e., CA1 PNs, whose soma are located in stratum radiatum. These data provide insight into local hippocampal circuitry and reveal how localized plasticity can potentially control information flow in the larger hippocampal network.

Memory circuits: CA2.

The hippocampus is a central region in the coding of spatial, temporal and episodic memory. Recent discoveries have revealed surprising and complex roles of the small area CA2 in hippocampal function. Lesion studies have revealed that this region is required for social memory formation. Area CA2 is targeted by extra-hippocampal paraventricular inputs that release vasopressin and can act to enhance social memory performance. In vivo recordings have revealed nonconventional activity by neurons in this region that act to both initiate hippocampal sharp-wave ripple events as well as encode spatial information during immobility. Silencing of CA2 pyramidal neurons has revealed that this area also acts to control hippocampal network excitability during encoding, and this balance of excitation and inhibition is disrupted in disease. This review summarizes recent findings and attempts to integrate these results into pre-existing models.

Hippocampal area CA2: properties and contribution to hippocampal function.

This review focuses on area CA2 of the hippocampus, as recent results have revealed the unique properties and surprising role of this region in encoding social, temporal and contextual aspects of memory. Originally identified and described by Lorente de No, in 1934, this region of the hippocampus has unique intra-and extra-hippocampal connectivity, sending and receiving input to septal and hypothalamic regions. Recent in vivo studies have indicated that CA2 pyramidal neurons encode spatial information during immobility and play an important role in the generation of sharp-wave ripples. Furthermore, CA2 neurons act to control overall excitability in the hippocampal network and have been found to be consistently altered in psychiatric diseases, indicating that normal function of this region is necessary for normal cognition. With its unique role, area CA2 has a unique molecular profile, interneuron density and composition. Furthermore, this region has an unusual manifestation of synaptic plasticity that does not occur post-synaptically at pyramidal neuron dendrities but through the local network of inhibitory neurons. While much progress has recently been made in understanding the large contribution of area CA2 to social memory formation, much still needs to be learned.

Heterogeneity in Kv2 Channel Expression Shapes Action Potential Characteristics and Firing Patterns in CA1 versus CA2 Hippocampal Pyramidal Neurons.

The CA1 region of the hippocampus plays a critical role in spatial and contextual memory, and has well-established circuitry, function and plasticity. In contrast, the properties of the flanking CA2 pyramidal neurons (PNs), important for social memory, and lacking CA1-like plasticity, remain relatively understudied. In particular, little is known regarding the expression of voltage-gated K+ (Kv) channels and the contribution of these channels to the distinct properties of intrinsic excitability, action potential (AP) waveform, firing patterns and neurotransmission between CA1 and CA2 PNs. In the present study, we used multiplex fluorescence immunolabeling of mouse brain sections, and whole-cell recordings in acute mouse brain slices, to define the role of heterogeneous expression of Kv2 family Kv channels in CA1 versus CA2 pyramidal cell excitability. Our results show that the somatodendritic delayed rectifier Kv channel subunits Kv2.1, Kv2.2, and their auxiliary subunit AMIGO-1 have region-specific differences in expression in PNs, with the highest expression levels in CA1, a sharp decrease at the CA1-CA2 boundary, and significantly reduced levels in CA2 neurons. PNs in CA1 exhibit a robust contribution of Guangxitoxin-1E-sensitive Kv2-based delayed rectifier current to AP shape and after-hyperpolarization potential (AHP) relative to that seen in CA2 PNs. Our results indicate that robust Kv2 channel expression confers a distinct pattern of intrinsic excitability to CA1 PNs, potentially contributing to their different roles in hippocampal network function.

Chronic Loss of CA2 Transmission Leads to Hippocampal Hyperexcitability.

Hippocampal CA2 pyramidal cells project into both the neighboring CA1 and CA3 subfields, leaving them well positioned to influence network physiology and information processing for memory and space. While recent work has suggested unique roles for CA2, including encoding position during immobility and generating ripple oscillations, an interventional examination of the integrative functions of these connections has yet to be reported. Here we demonstrate that CA2 recruits feedforward inhibition in CA3 and that chronic genetically engineered shutdown of CA2-pyramidal-cell synaptic transmission consequently results in increased excitability of the recurrent CA3 network. In behaving mice, this led to spatially triggered episodes of network-wide hyperexcitability during exploration accompanied by the emergence of high-frequency discharges during rest. These findings reveal CA2 as a regulator of network processing in hippocampus and suggest that CA2-mediated inhibition in CA3 plays a key role in establishing the dynamic excitatory and inhibitory balance required for proper network function.

Hippocampal Area CA2: An Overlooked but Promising Therapeutic Target.

While the hippocampus has long been recognized as a brain structure specialized in mapping 'space' in rodents, human studies and now recent data from rodents have shown that its function extends well beyond spatial coding. Recently, an overlooked area of the hippocampus, CA2, has emerged as a critical region for social memory. This area is also uniquely altered during several pathologies such as schizophrenia and age-related dementia. Because of its singular connectivity, we propose that area CA2 resides at the interface between emotional brain activity and higher cognitive function. Furthermore, because of the unique expression of multiple neuromodulator receptors in area CA2, we posit that this region may represent a fruitful therapeutic target for diseases where social dysfunction occurs.

Age-Dependent Specific Changes in Area CA2 of the Hippocampus and Social Memory Deficit in a Mouse Model of the 22q11.2 Deletion Syndrome.

Several neuropsychiatric disorders are associated with cognitive and social dysfunction. Postmortem studies of patients with schizophrenia have revealed specific changes in area CA2, a long-overlooked region of the hippocampus recently found to be critical for social memory formation. To examine how area CA2 is altered in psychiatric illness, we used the Df(16)A(+/-) mouse model of the 22q11.2 microdeletion, a genetic risk factor for developing several neuropsychiatric disorders, including schizophrenia. We report several age-dependent CA2 alterations: a decrease in the density of parvalbumin-expressing interneurons, a reduction in the amount of feedforward inhibition, and a change in CA2 pyramidal-neuron intrinsic properties. Furthermore, we found that area CA2 is less plastic in Df(16)A(+/-) mice, making it nearly impossible to evoke action potential firing in CA2 pyramidal neurons. Finally, we show that Df(16)A(+/-) mice display impaired social cognition, providing a potential mechanism and a neural substrate for this impairment in psychiatric disorders.

Inhibitory Plasticity Permits the Recruitment of CA2 Pyramidal Neurons by CA3

Area CA2 is emerging as an important region for hippocampal memory formation. However, how CA2 pyramidal neurons (PNs) are engaged by intrahippocampal inputs remains unclear. Excitatory transmission between CA3 and CA2 is strongly inhibited and is not plastic. We show in mice that different patterns of activity can in fact increase the excitatory drive between CA3 and CA2. We provide evidence that this effect is mediated by a long-term depression at inhibitory synapses (iLTD), as it is evoked by the same protocols and shares the same pharmacology. In addition, we show that the net excitatory drive of distal inputs is also increased after iLTD induction. The disinhibitory increase in excitatory drive is sufficient to allow CA3 inputs to evoke action potential firing in CA2 PNs. Thus, these data reveal that the output of CA2 PNs can be gated by the unique activity-dependent plasticity of inhibitory neurons in area CA2.

Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes.

Specification of cell identity during development depends on exposure of cells to sequences of extrinsic cues delivered at precise times and concentrations. Identification of combinations of patterning molecules that control cell fate is essential for the effective use of human pluripotent stem cells (hPSCs) for basic and translational studies. Here we describe a scalable, automated approach to systematically test the combinatorial actions of small molecules for the targeted differentiation of hPSCs. Applied to the generation of neuronal subtypes, this analysis revealed an unappreciated role for canonical Wnt signaling in specifying motor neuron diversity from hPSCs and allowed us to define rapid (14 days), efficient procedures to generate spinal and cranial motor neurons as well as spinal interneurons and sensory neurons. Our systematic approach to improving hPSC-targeted differentiation should facilitate disease modeling studies and drug screening assays.

Delta-opioid receptors mediate unique plasticity onto parvalbumin-expressing interneurons in area CA2 of the hippocampus.

Inhibition is critical for controlling information transfer in the brain. However, the understanding of the plasticity and particular function of different interneuron subtypes is just emerging. Using acute hippocampal slices prepared from adult mice, we report that in area CA2 of the hippocampus, a powerful inhibitory transmission is acting as a gate to prevent CA3 inputs from driving CA2 neurons. Furthermore, this inhibition is highly plastic, and undergoes a long-term depression following high-frequency 10 Hz or theta-burst induction protocols. We describe a novel form of long-term depression at parvalbumin-expressing (PV+) interneuron synapses that is dependent on delta-opioid receptor (DOR) activation. Additionally, PV+ interneuron transmission is persistently depressed by DOR activation in area CA2 but only transiently depressed in area CA1. These results provide evidence for a differential temporal modulation of PV+ synapses between two adjacent cortical circuits, and highlight a new function of PV+ cells in controlling information transfer.

Synaptic integration by different dendritic compartments of hippocampal CA1 and CA2 pyramidal neurons.

Pyramidal neurons have a complex dendritic arbor containing tens of thousands of synapses. In order for the somatic/axonal membrane potential to reach action potential threshold, concurrent activation of multiple excitatory synapses is required. Frequently, instead of a simple algebraic summation of synaptic potentials in the soma, different dendritic compartments contribute to the integration of multiple inputs, thus endowing the neuron with a powerful computational ability. Most pyramidal neurons share common functional properties. However, different and sometimes contrasting dendritic integration rules are also observed. In this review, we focus on the dendritic integration of two neighboring pyramidal neurons in the hippocampus: the well-characterized CA1 and the much less understood CA2. The available data reveal that the dendritic integration of these neurons is markedly different even though they are targeted by common inputs at similar locations along their dendrites. This contrasting dendritic integration results in different routing of information flow and generates different corticohippocampal loops.

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum.

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites.

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 aa domain in the conserved central core of TRIP8b; and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.

Increased seizure severity and seizure-related death in mice lacking HCN1 channels.

Persistent down-regulation in the expression of the hyperpolarization-activated HCN1 cation channel, a key determinant of intrinsic neuronal excitability, has been observed in febrile seizure, temporal lobe epilepsy, and generalized epilepsy animal models, as well as in patients with epilepsy. However, the role and importance of HCN1 down-regulation for seizure activity is unclear. To address this question we determined the susceptibility of mice with either a general or forebrain-restricted deletion of HCN1 to limbic seizure induction by amygdala kindling or pilocarpine administration. Loss of HCN1 expression in both mouse lines is associated with higher seizure severity and higher seizure-related mortality, independent of the seizure-induction method used. Therefore, down-regulation of HCN1 associated with human epilepsy and rodent models may be a contributing factor in seizure behavior.

TRIP8b splice variants form a family of auxiliary subunits that regulate gating and trafficking of HCN channels in the brain.

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

Relationship between pore occupancy and gating in BK potassium channels.

Permeant ions can have significant effects on ion channel conformational changes. To further understand the relationship between ion occupancy and gating conformational changes, we have studied macroscopic and single-channel gating of BK potassium channels with different permeant monovalent cations. While the slopes of the conductance-voltage curve were reduced with respect to potassium for all permeant ions, BK channels required stronger depolarization to open only when thallium was the permeant ion. Thallium also slowed the activation and deactivation kinetics. Both the change in kinetics and the shift in the GV curve were dependent on the thallium passing through the permeation pathway, as well as on the concentration of thallium. There was a decrease in the mean open time and an increase in the number of short flicker closing events with thallium as the permeating ion. Mean closed durations were unaffected. Application of previously established allosteric gating models indicated that thallium specifically alters the opening and closing transition of the channel and does not alter the calcium activation or voltage activation pathways. Addition of a closed flicker state into the allosteric model can account for the effect of thallium on gating. Consideration of the thallium concentration dependence of the gating effects suggests that the flicker state may correspond to the collapsed selectivity filter seen in crystal structures of the KcsA potassium channel under the condition of low permeant ion concentration.