Hippocampus shapes cortical sensory output and novelty coding through a direct feedback circuit.

To extract behaviorally relevant information from our surroundings, our brains constantly integrate and compare incoming sensory information with those stored as memories. Cortico-hippocampal interactions could mediate such interplay between sensory processing and memory recall1-4 but this remains to be demonstrated. Recent work parsing entorhinal cortex-to-hippocampus circuitry show its role in episodic memory formation5-7 and spatial navigation8. However, the organization and function of the hippocampus-to-cortex back-projection circuit remains uncharted. We combined circuit mapping, physiology and behavior with optogenetic manipulations, and computational modeling to reveal how hippocampal feedback modulates cortical sensory activity and behavioral output. Here we show a new direct hippocampal projection to entorhinal cortex layer 2/3, the very layer that projects multisensory input to the hippocampus. Our finding challenges the canonical cortico-hippocampal circuit model where hippocampal feedback only reaches entorhinal cortex layer 2/3 indirectly via layer 5. This direct hippocampal input integrates with cortical sensory inputs in layer 2/3 neurons to drive their plasticity and spike output, and provides an important novelty signal during behavior for coding objects and their locations. Through the sensory-memory feedback loop, hippocampus can update real-time cortical sensory processing, efficiently and iteratively, thereby imparting the salient context for adaptive learned behaviors with new experiences.

Brain Abnormalities in Patients with Germline Variants in H3F3: Novel Imaging Findings and Neurologic Symptoms Beyond Somatic Variants and Brain Tumors.

BACKGROUND AND PURPOSE: Pathogenic somatic variants affecting the genes Histone 3 Family 3A and 3B (H3F3) are extensively linked to the process of oncogenesis, in particular related to central nervous system tumors in children. Recently, H3F3 germline missense variants were described as the cause of a novel pediatric neurodevelopmental disorder. We aimed to investigate patterns of brain MR imaging of individuals carrying H3F3 germline variants. MATERIALS AND METHODS: In this retrospective study, we included individuals with proved H3F3 causative genetic variants and available brain MR imaging scans. Clinical and demographic data were retrieved from available medical records. Molecular genetic testing results were classified using the American College of Medical Genetics criteria for variant curation. Brain MR imaging abnormalities were analyzed according to their location, signal intensity, and associated clinical symptoms. Numeric variables were described according to their distribution, with median and interquartile range. RESULTS: Eighteen individuals (10 males, 56%) with H3F3 germline variants were included. Thirteen of 18 individuals (72%) presented with a small posterior fossa. Six individuals (33%) presented with reduced size and an internal rotational appearance of the heads of the caudate nuclei along with an enlarged and squared appearance of the frontal horns of the lateral ventricles. Five individuals (28%) presented with dysgenesis of the splenium of the corpus callosum. Cortical developmental abnormalities were noted in 8 individuals (44%), with dysgyria and hypoplastic temporal poles being the most frequent presentation. CONCLUSIONS: Imaging phenotypes in germline H3F3-affected individuals are related to brain features, including a small posterior fossa as well as dysgenesis of the corpus callosum, cortical developmental abnormalities, and deformity of lateral ventricles.

Transgenic silencing of neurons in the mammalian brain by expression of the allatostatin receptor (AlstR).

The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting "silencer" transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K(+) channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

Abolition of long-term stability of new hippocampal place cell maps by NMDA receptor blockade.

Hippocampal pyramidal cells are called place cells because each cell tends to fire only when the animal is in a particular part of the environment-the cell's firing field. Acute pharmacological blockade of N-methyl-D-aspartate (NMDA) glutamate receptors was used to investigate how NMDA-based synaptic plasticity participates in the formation and maintenance of the firing fields. The results suggest that the formation and short-term stability of firing fields in a new environment involve plasticity that is independent of NMDA receptor activation. By contrast, the long-term stabilization of newly established firing fields required normal NMDA receptor function and, therefore, may be related to other NMDA-dependent processes such as long-term potentiation and spatial learning.

K+ channel subunit isoforms with divergent carboxy-terminal sequences carry distinct membrane targeting signals.

Kv3 K+ channel genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K+ channel proteins that differ only in their C-termini. This divergence does not affect the electrophysiological properties of the channels expressed by these proteins. A similar alternative splicing with unknown function is seen in K+ channel genes of other families. We have investigated the possibility that the alternative splicing serves to generate channel subunits with different membrane targeting signals by examining the sorting behavior of three alternatively-spliced Kv3.2 isoforms when expressed in polarized MDCK cells. Two Kv3.2 proteins, Kv3.2b and Kv3.2c were expressed predominantly in the apical membrane, while Kv3.2a was localized mainly to the basolateral side (thought to be equivalent to the axonal and somatodendritic compartments in neurons, respectively). The Kv3.2 mRNA transcripts used in these studies are identical except for their 3' sequence, encoding the extreme C-terminal domain of the protein and the 3'UTR of the mRNA. However, the proteins achieve the same localizations in MDCK cells when expressed from constructs containing or lacking the 3'UTR, indicating that the differential localization is due to targeting signals present in the C' terminal domain of the protein. These results suggest that the alternative splicing of Kv3 genes is involved in channel localization. Since the precise localization of any given ion channel on the neuronal surface has significant functional implications, the results shown here suggest an important function for the alternative splicing of 3' ends seen in many K+ channel genes.

G-protein-gated inward rectifier K+ channel proteins (GIRK1) are present in the soma and dendrites as well as in nerve terminals of specific neurons in the brain.

G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors.

Thalamocortical projections have a K+ channel that is phosphorylated and modulated by cAMP-dependent protein kinase.

The finding that some K+ channel mRNAs are restricted to certain populations of neurons in the CNS suggests that there are K+ channels tailored to certain neuronal circuits. One such example are the transcripts from the KV3.2 gene, the majority of which are expressed in thalamic relay neurons. To gain insights into the specific roles of KV3.2 subunits, site specific antibodies were raised to determine their localization in thalamic relay neurons. Immunohistochemical and focal lesioning studies demonstrate that KV3.2 proteins are localized to the terminal fields of thalamocortical projections. It is also shown that KV3.2 channels expressed in vitro are strongly inhibited through phosphorylation by cAMP-dependent protein kinase (PKA). Channels containing KV3.1 subunits, which otherwise exhibit nearly identical electrophysiological properties in heterologous expression systems but have a different and less restricted pattern of expression in the CNS, are not affected by PKA. Therefore, this modulation might be associated with the specific roles of KV3.2 subunits. Furthermore, we demonstrate that KV3.2 proteins can be phosphorylated in situ by intrinsic PKA. KV3.2 subunits display properties and have a localization consistent with a role in the regulation of the efficacy of the thalamocortical synapse, and could thereby participate in the neurotransmitter-mediated control of functional states of the thalamocortical system associated with global states of awareness.

Identification of molecular components of A-type channels activating at subthreshold potentials.

1. Xenopus oocytes injected with rat brain mRNA express a transient K+ current similar to the A current that activates transiently near the threshold for Na+ action potential generation (ISA) seen in somatic recordings from neurons. We used hybrid arrest with antisense oligonucleotides to investigate which of the cloned K+ channel proteins might be components of the channels responsible for the ISA expressed from brain mRNA. An oligonucleotide complementary to a sequence common to all known mammalian Shal-related mRNAs [KV4.1, KV4.2, and KV4.3 (the nomenclature of Sh K+ channel genes of Chandy and colleagues was used in this paper)] blocked the expression of the ISA. An oligonucleotide complementary only to the KV4.2 mRNA, the most abundant Shal-related transcript in rat brain RNA preparations, was also quite efficient in arresting the expression of the ISA from brain. These experiments indicate that Shal-related proteins are important components of the channels carrying the ISA expressed in oocytes injected with brain mRNA. However, there are several significant differences between this ISA and the currents expressed in the same oocytes by in vitro transcribed KV4.1 or KV4.2 cRNA. Most of these differences are eliminated if KV4.1 or KV4.2 cRNA is coinjected with brain poly-(A) RNA treated with antisense oligonucleotides which arrest the expression of the ISA, or with a 2-4Kb rat brain poly-(A) RNA fraction which does not express detectable K+ currents under the same recording conditions. These data support the hypothesis that ISA channels such as those expressed from brain mRNA contain Shal proteins that can be modified by proteins encoded in RNAs that by themselves do not express K+ currents.

Differential expression of Shaw-related K+ channels in the rat central nervous system.

The family of mammalian genes related to the Drosophila Shaker gene, consisting of four subfamilies, is thought to encode subunits of tetrameric voltage-gated K+ channels. There is compelling evidence that subunits of the same subfamily, but not of different subfamilies, form heteromultimeric channels in vitro, and thus, each gene subfamily is postulated to encode components of an independent channel system. In order to identify cells with native channels containing subunits of one of these subfamilies (Shaw-related or ShIII), the cellular distribution of ShIII transcripts was examined by Northern blot analysis and in situ hybridization. Three of four ShIII genes (KV3.1, KV3.2, and KV3.3) are expressed mainly in the CNS. KV3.4 transcripts are also present in the CNS but are more abundant in skeletal muscle. In situ hybridization studies in the CNS reveal discrete and specific neuronal populations that prominently express ShIII mRNAs, both in projecting and in local circuit neurons. In the cerebral cortex, hippocampus, and caudate-putamen, subsets of neurons can be distinguished by the expression of specific ShIII mRNAs. Each ShIII gene exhibits a unique pattern of expression; however, many neuronal populations expressing KV3.1 transcripts also express KV3.3 mRNAs. Furthermore, KV3.4 transcripts are present, albeit at lower levels, in several of the neuronal populations that also express KV3.1 and/or KV3.3 mRNAs, revealing a high potential for heteromultimer formation between the products of three of the four genes. Expression of ShIII cRNAs in Xenopus oocytes was used to explore the functional consequences of heteromultimer formation between ShIII subunits. Small amounts of KV3.4 cRNA, which expresses small, fast-inactivating currents when injected alone, produced fast-inactivating currents that are severalfold larger when coinjected with an excess of KV3.1 or KV3.3 cRNA. This amplification is due to both an increase in single-channel conductance in the heteromultimeric channels and the observation that less than four, perhaps even a single KV3.4 subunit is sufficient to impart fast-inactivating properties to the channel. The oocyte experiments indicate that the apparently limited, low-level expression of KV3.4 in the CNS is potentially significant. The anatomical studies suggest that heteromultimer formation between ShIII proteins might be a common feature in the CNS. Moreover, the possibility that the subunit composition of heteromultimers varies in different neurons should be considered, since the ratios of overlapping signals change from one neuronal population to another. In order to proceed with functional analysis of native ShIII channels, it is important to known which subunit compositions might occur in vivo. The studies presented here provide important clues for the identification of native homo- and heteromultimeric ShIII channels in neurons.

Region-specific expression of a K+ channel gene in brain.

Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions.

Cloning of ShIII (Shaw-like) cDNAs encoding a novel high-voltage-activating, TEA-sensitive, type-A K+ channel.

Transient voltage-dependent potassium (K+) currents, also known as A currents, have been of great interest to neurophysiologists due to their special roles in neuronal excitability. Several cDNAs encoding transcripts expressing A currents have been characterized. Recently, a cDNA (KShIIIC or Raw3) was isolated which expresses an unusual A current that is highly sensitive to TEA, and activates at potentials more positive than -20 mV. Channels containing this protein may have specialized roles in modulating the electrical behaviour of neurons. Here we report the isolation and characterization of two rat cDNAs corresponding to two alternatively spliced transcripts (KShIIID.1 and KShIIID.2) from another gene (KShIIID) of the same subfamily as KShIIIC, the ShIII or Shaw-related gene subfamily. KShIIID.1 also expresses an unusual high-voltage-activating, TEA-sensitive A-type channel. There are, however, significant differences between KShIIIC and KShIIID channels which may have interesting functional consequences. The two most important differences are: (i) KShIIID channels conduct in the steady-state over a much broader window of potentials than KShIIIC; this reflects differences between the kinetic schemes of the two channels; and (ii) KShIIID inactivates with significantly slower kinetics than KShIIIC. The identification of KShIIID transcripts contributes to our knowledge of the molecular components that may determine the functional diversity of A currents and provides exciting opportunities to increase our understanding of the structure and function of K+ channels.

Identification of an unusual deletion within homologous repeats of human reticulocyte beta-spectrin and probable peptide polymorphism.

We screened two different human reticulocyte cDNA libraries with beta-spectrin(beta Sp)-specific polyclonal antibodies and with our original radiolabeled human BSP cDNA probe (encoding beta Sp). Of the 20 independent clones, the largest had about a 2.5-kb insert corresponding to the deduced amino acid (aa) sequence of the beta-7 to beta-14 repetitive segments. Among these segments, segment 12 was 7 aa shorter than the other repetitive segments. We showed that this truncation was not a result of (i) cloning artifact, (ii) alternate splicing, or (iii) common genomic polymorphism by additional examination of 14 individual human chromosomes. Recently, another laboratory described the BSP nucleotide (nt) sequence overlapping partially with our sequence. These overlapping sequences were homologous with the exception of two nt differences at the positions 1342 and 1514. The discrepancy at nt 1342 changes the His to Arg. This newly derived probe has been used to find an additional example of BSP restriction fragment length polymorphism.