An update to Hippocampome.org by integrating single-cell phenotypes with circuit function in vivo.

Understanding brain operation demands linking basic behavioral traits to cell-type specific dynamics of different brain-wide subcircuits. This requires a system to classify the basic operational modes of neurons and circuits. Single-cell phenotyping of firing behavior during ongoing oscillations in vivo has provided a large body of evidence on entorhinal-hippocampal function, but data are dispersed and diverse. Here, we mined literature to search for information regarding the phase-timing dynamics of over 100 hippocampal/entorhinal neuron types defined in Hippocampome.org. We identified missing and unresolved pieces of knowledge (e.g., the preferred theta phase for a specific neuron type) and complemented the dataset with our own new data. By confronting the effect of brain state and recording methods, we highlight the equivalences and differences across conditions and offer a number of novel observations. We show how a heuristic approach based on oscillatory features of morphologically identified neurons can aid in classifying extracellular recordings of single cells and discuss future opportunities and challenges towards integrating single-cell phenotypes with circuit function.

Proximodistal structure of theta coordination in the dorsal hippocampus of epileptic rats.

Coherent neuronal activity in the hippocampal-entorhinal circuit is a critical mechanism for episodic memory function, which is typically impaired in temporal lobe epilepsy. To better understand how this mechanism is implemented and degraded in this condition, we used normal and epileptic rats to examine theta activity accompanying active exploration. Assisted by multisite recordings of local field potentials (LFPs) and layer-specific profiling of input pathways, we provide detailed quantification of the proximodistal coherence of theta activity in the dorsal hippocampus of these animals. Normal rats showed stronger coordination between the temporoammonic and perforant entorhinal inputs (measured from lamina-specific current source density signals) at proximal locations, i.e., closer to CA3; while epileptic rats exhibited stronger interactions at distal locations, i.e., closer to subiculum. This opposing trend in epileptic rats was associated with the reorganization of the temporoammonic and perforant pathways that accompany hippocampal sclerosis, the pathological hallmark of this disease. In addition to this connectivity constraint, we discovered that the appropriate timing between entorhinal inputs arriving over several theta cycles at the proximal and distal ends of the dorsal hippocampus was impaired in epileptic rats. Computational reconstruction of LFP signals predicted that restoring timing variability has a major impact on repairing theta coherence. This manipulation, when tested pharmacologically via systemic administration of group III mGluR antagonists, successfully re-established theta coordination of LFPs in epileptic rats. Thus, proximodistal organization of entorhinal inputs is instrumental in temporal lobe physiology and a candidate mechanism to study cognitive comorbidities of temporal lobe epilepsy.

Specific impairment of "what-where-when" episodic-like memory in experimental models of temporal lobe epilepsy.

Episodic memory deficit is a common cognitive disorder in human temporal lobe epilepsy (TLE). However, no animal model of TLE has been shown to specifically replicate this cognitive dysfunction, which has limited its translational appeal. Here, using a task that tests for nonverbal correlates of episodic-like memory in rats, we show that kainate-treated TLE rats exhibit a selective impairment of the "what-where-when" memory while preserving other forms of hippocampal-dependent memories. Assisted by multisite silicon probes, we recorded from the dorsal hippocampus of behaving animals to control for seizure-related factors and to look for electrophysiological signatures of cognitive impairment. Analyses of hippocampal local field potentials showed that both the power of theta rhythm and its coordination across CA1 and the DG-measured as theta coherence and phase locking-were selectively disrupted. This disruption represented a basal condition of the chronic epileptic hippocampus that was linked to different features of memory impairment. Theta power was more correlated with the spatial than with the temporal component of the task, while measures of theta coordination correlated with the temporal component. We conclude that episodic-like memory, as tested in the what-where-when task, is specifically affected in experimental TLE and that the impairment of hippocampal theta activity might be central to this dysfunction.

Immunohistochemical field parcellation of the human hippocampus along its antero-posterior axis.

The primate hippocampus includes the dentate gyrus, cornu ammonis (CA), and subiculum. CA is subdivided into four fields (CA1-CA3, plus CA3h/hilus of the dentate gyrus) with specific pyramidal cell morphology and connections. Work in non-human mammals has shown that hippocampal connectivity is precisely patterned both in the laminar and longitudinal axes. One of the main handicaps in the study of neuropathological semiology in the human hippocampus is the lack of clear laminar and longitudinal borders. The aim of this study was to explore a histochemical segmentation of the adult human hippocampus, integrating field (medio-lateral), laminar, and anteroposterior longitudinal patterning. We provide criteria for head-body-tail field and subfield parcellation of the human hippocampus based on immunodetection of Rabphilin3a (Rph3a), Purkinje-cell protein 4 (PCP4), Chromogranin A and Regulation of G protein signaling-14 (RGS-14). Notably, Rph3a and PCP4 allow to identify the border between CA3 and CA2, while Chromogranin A and RGS-14 give specific staining of CA2. We also provide novel histological data about the composition of human-specific regions of the anterior and posterior hippocampus. The data are given with stereotaxic coordinates along the longitudinal axis. This study provides novel insights for a detailed region-specific parcellation of the human hippocampus useful for human brain imaging and neuropathology.

Topological analysis of sharp-wave ripple waveforms reveals input mechanisms behind feature variations.

The reactivation of experience-based neural activity patterns in the hippocampus is crucial for learning and memory. These reactivation patterns and their associated sharp-wave ripples (SWRs) are highly variable. However, this variability is missed by commonly used spectral methods. Here, we use topological and dimensionality reduction techniques to analyze the waveform of ripples recorded at the pyramidal layer of CA1. We show that SWR waveforms distribute along a continuum in a low-dimensional space, which conveys information about the underlying layer-specific synaptic inputs. A decoder trained in this space successfully links individual ripples with their expected sinks and sources, demonstrating how physiological mechanisms shape SWR variability. Furthermore, we found that SWR waveforms segregated differently during wakefulness and sleep before and after a series of cognitive tasks, with striking effects of novelty and learning. Our results thus highlight how the topological analysis of ripple waveforms enables a deeper physiological understanding of SWRs.

Aberrant survival of hippocampal Cajal-Retzius cells leads to memory deficits, gamma rhythmopathies and susceptibility to seizures in adult mice.

Cajal-Retzius cells (CRs) are transient neurons, disappearing almost completely in the postnatal neocortex by programmed cell death (PCD), with a percentage surviving up to adulthood in the hippocampus. Here, we evaluate CR's role in the establishment of adult neuronal and cognitive function using a mouse model preventing Bax-dependent PCD. CRs abnormal survival resulted in impairment of hippocampus-dependent memory, associated in vivo with attenuated theta oscillations and enhanced gamma activity in the dorsal CA1. At the cellular level, we observed transient changes in the number of NPY+ cells and altered CA1 pyramidal cell spine density. At the synaptic level, these changes translated into enhanced inhibitory currents in hippocampal pyramidal cells. Finally, adult mutants displayed an increased susceptibility to lethal tonic-clonic seizures in a kainate model of epilepsy. Our data reveal that aberrant survival of a small proportion of postnatal hippocampal CRs results in cognitive deficits and epilepsy-prone phenotypes in adulthood.

Emergent dynamics of fast ripples in the epileptic hippocampus.

Fast ripples are a type of transient high-frequency oscillations recorded from the epileptogenic regions of the hippocampus and the temporal cortex of epileptic humans and rodents. These events presumably reflect hypersynchronous bursting of pyramidal cells. However, the oscillatory spectral content of fast ripples varies from 250 to 800 Hz, well above the maximal firing frequency of most hippocampal pyramidal neurons. How such high-frequency oscillations are generated is therefore unclear. Here, we combine computational simulations of fast ripples with multisite and juxtacellular recordings in vivo to examine the underlying mechanisms in the hippocampus of epileptic rats. We show that populations of bursting cells firing individually at 100-400 Hz can create fast ripples according to two main firing regimes: (1) in-phase synchronous firing resulting in "pure" fast ripples characterized by single spectral peaks that reflect single-cell behavior and (2) out-of-phase firing that results in "emergent" fast ripples. Using simulations, we found that fast ripples generated under these two different regimes can be quantitatively separated by their spectral characteristics, and we took advantage of this separability to examine their dynamics in vivo. We found that in-phase firing can reach frequencies up to 300 Hz in the CA1 and up to 400 Hz in the dentate gyrus. The organization of out-of-phase firing is determined by firing delays between cells discharging at low frequencies. The two firing regimes compete dynamically, alternating randomly from one fast ripple event to the next, and they reflect the functional dynamic organization of the different regions of the hippocampus.

Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis.

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.

Prox1 expression in rod precursors and Müller cells.

The transcription factor Prox1 acts in rodent retinogenesis, at least in promoting cell cycle withdrawal and horizontal cell production. In the mature retina, this protein is detected at the inner nuclear layer of all vertebrate groups. We have made a neurochemical characterisation of Prox1(+) cell types in two different vertebrate groups: mammals and fish. As well as Prox1(+) horizontal cells, we have observed Prox1(+)/PKC-alpha(+) rod bipolar cells in mouse and cone ON and mixed b bipolar cells in goldfish. In mouse, only some CB(+) and CR(+) amacrine cells are Prox1(+) and the TH(+) and CR(+) amacrine cells are Prox1(-). However, in goldfish all CR(+) amacrine cells and TH(+) interplexiform cells are Prox1(+) and in the GCL displaced amacrine cells are also Prox1(+). Besides its expression in different interneuron subpopulations, we demonstrate, for the first time, the presence of Prox1 in the GS(+) and CRALBP(+) Müller cells in the retina of adult mammals and in developing and mature retina of fish. The presence of Prox1 in these cells appears to be related to survival or maintenance of their phenotype. We also demonstrate that in fish, where retinal formation persists into adulthood, Prox1 is expressed in dividing PCNA(+) cells at the peripheral growing zone, in rod progenitors at the inner and outer nuclear layers as well as in early progenitors during a retinal regeneration process after cryo-lesion of the peripheral growing zone. Therefore, Prox1 functions in vertebrate retinogenesis may be more complex than previously expected.

Multimodal determinants of phase-locked dynamics across deep-superficial hippocampal sublayers during theta oscillations.

Theta oscillations play a major role in temporarily defining the hippocampal rate code by translating behavioral sequences into neuronal representations. However, mechanisms constraining phase timing and cell-type-specific phase preference are unknown. Here, we employ computational models tuned with evolutionary algorithms to evaluate phase preference of individual CA1 pyramidal cells recorded in mice and rats not engaged in any particular memory task. We applied unbiased and hypothesis-free approaches to identify effects of intrinsic and synaptic factors, as well as cell morphology, in determining phase preference. We found that perisomatic inhibition delivered by complementary populations of basket cells interacts with input pathways to shape phase-locked specificity of deep and superficial pyramidal cells. Somatodendritic integration of fluctuating glutamatergic inputs defined cycle-by-cycle by unsupervised methods demonstrated that firing selection is tuneable across sublayers. Our data identify different mechanisms of phase-locking selectivity that are instrumental for flexible dynamical representations of theta sequences.

Methods for single-cell recording and labeling in vivo.

Targeting individual neurons in vivo is a key method to study the role of single cell types within local and brain-wide microcircuits. While novel technological developments now permit assessing activity from large number of cells simultaneously, there is currently no better solution than glass micropipettes to relate the physiology and morphology of single-cells. Sharp intracellular, juxtacellular, loose-patch and whole-cell approaches are some of the configurations used to record and label individual neurons. Here, we review procedures to establish successful electrophysiological recordings in vivo followed by appropriate labeling for post hoc morphological analysis. We provide operational recommendations for optimizing each configuration and a generic framework for functional, neurochemical and morphological identification of the different cell-types in a given region. Finally, we highlight emerging approaches that are challenging our current paradigms for single-cell recording and labeling in the living brain.

Proximodistal Organization of the CA2 Hippocampal Area.

The proximodistal axis is considered a major organizational principle of the hippocampus. At the interface between the hippocampus and other brain structures, CA2 apparently breaks this rule. The region is involved in social, temporal, and contextual memory function, but mechanisms remain elusive. Here, we reveal cell-type heterogeneity and a characteristic expression gradient of the transcription factor Sox5 within CA2 in the rat. Using intracellular and extracellular recordings followed by neurochemical identification of single cells, we find marked proximodistal trends of synaptic activity, subthreshold membrane potentials, and phase-locked firing coupled to theta and gamma oscillations. Phase-shifting membrane potentials and opposite proximodistal correlations with theta sinks and sources at different layers support influences from different current generators. CA2 oscillatory activity and place coding of rats running in a linear maze reflect proximodistal state-dependent trends. We suggest that the structure and function of CA2 are distributed along the proximodistal hippocampal axis.

Mechanisms for Selective Single-Cell Reactivation during Offline Sharp-Wave Ripples and Their Distortion by Fast Ripples.

Memory traces are reactivated selectively during sharp-wave ripples. The mechanisms of selective reactivation, and how degraded reactivation affects memory, are poorly understood. We evaluated hippocampal single-cell activity during physiological and pathological sharp-wave ripples using juxtacellular and intracellular recordings in normal and epileptic rats with different memory abilities. CA1 pyramidal cells participate selectively during physiological events but fired together during epileptic fast ripples. We found that firing selectivity was dominated by an event- and cell-specific synaptic drive, modulated in single cells by changes in the excitatory/inhibitory ratio measured intracellularly. This mechanism collapses during pathological fast ripples to exacerbate and randomize neuronal firing. Acute administration of a use- and cell-type-dependent sodium channel blocker reduced neuronal collapse and randomness and improved recall in epileptic rats. We propose that cell-specific synaptic inputs govern firing selectivity of CA1 pyramidal cells during sharp-wave ripples.

Altered Oscillatory Dynamics of CA1 Parvalbumin Basket Cells during Theta-Gamma Rhythmopathies of Temporal Lobe Epilepsy.

Recent reports in human demonstrate a role of theta-gamma coupling in memory for spatial episodes and a lack of coupling in people experiencing temporal lobe epilepsy, but the mechanisms are unknown. Using multisite silicon probe recordings of epileptic rats engaged in episodic-like object recognition tasks, we sought to evaluate the role of theta-gamma coupling in the absence of epileptiform activities. Our data reveal a specific association between theta-gamma (30-60 Hz) coupling at the proximal stratum radiatum of CA1 and spatial memory deficits. We targeted the microcircuit mechanisms with a novel approach to identify putative interneuronal types in tetrode recordings (parvalbumin basket cells in particular) and validated classification criteria in the epileptic context with neurochemical identification of intracellularly recorded cells. In epileptic rats, putative parvalbumin basket cells fired poorly modulated at the falling theta phase, consistent with weaker inputs from Schaffer collaterals and attenuated gamma oscillations, as evaluated by theta-phase decomposition of current-source density signals. We propose that theta-gamma interneuronal rhythmopathies of the temporal lobe are intimately related to episodic memory dysfunction in this condition.

Determinants of different deep and superficial CA1 pyramidal cell dynamics during sharp-wave ripples.

Sharp-wave ripples represent a prominent synchronous activity pattern in the mammalian hippocampus during sleep and immobility. GABAergic interneuronal types are silenced or fire during these events, but the mechanism of pyramidal cell (PC) participation remains elusive. We found opposite membrane polarization of deep (closer to stratum oriens) and superficial (closer to stratum radiatum) rat CA1 PCs during sharp-wave ripples. Using sharp and multi-site recordings in combination with neurochemical profiling, we observed a predominant inhibitory drive of deep calbindin (CB)-immunonegative PCs that contrasts with a prominent depolarization of superficial CB-immunopositive PCs. Biased contribution of perisomatic GABAergic inputs, together with suppression of CA2 PCs, may explain the selection of CA1 PCs during sharp-wave ripples. A deep-superficial gradient interacted with behavioral and spatial effects to determine cell participation during sleep and awake sharp-wave ripples in freely moving rats. Thus, the firing dynamics of hippocampal PCs are exquisitely controlled at subcellular and microcircuit levels in a cell type-selective manner.

The glial design of a teleost optic nerve head supporting continuous growth.

This study demonstrates the peculiarities of the glial organization of the optic nerve head (ONH) of a fish, the tench (Tinca tinca), by using immunohistochemistry and electron microscopy. We employed antibodies specific for the macroglial cells: glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), and S100. We also used the N518 antibody to label the new ganglion cells' axons, which are continuously added to the fish retina, and the anti-proliferating cell nuclear antigen (PCNA) antibody to specifically locate dividing cells. We demonstrate a specific regional adaptation of the GS-S100-positive Müller cells' vitreal processes around the optic disc, strongly labeled with the anti-GFAP antibody. In direct contact with these Müller cells' vitreal processes, there are S100-positive astrocytes and S100-negative cells ultrastructurally identified as microglial cells. Moreover, a population of PCNA-positive cells, characterized as glioblasts, forms the limit between the retina and the optic nerve in a region homologous to the Kuhnt intermediary tissue of mammals. Finally, in the intraocular portion of the optic nerve there are differentiating oligodendrocytes arranged in rows. Both the glioblasts and the rows of developing cells could serve as a pool of glial elements for the continuous growth of the visual system.

Neuron-specific enolase, nucleotides, nucleosides, purine bases, oxypurines and uric acid concentrations in cerebrospinal fluid of children with meningitis.

To determine the effects of meningitis on cerebral energy metabolism, cerebrospinal fluid concentrations of adenosine monophosphate, inosine monophosphate, inosine, adenosine, guanosine, adenine, guanine, hypoxanthine, xanthine and urate were determined by high-performance liquid chromatography, and neuron-specific enolase by an enzyme immunoassay method, in 100 children with meningitis (45 bacterial, 46 viral and nine tuberculous), aged between 1 month and 13 years, and in 160 age-matched controls. Compared with controls, patients with bacterial meningitis showed high concentrations of hypoxanthine, xanthine and urate; patients with viral meningitis showed high concentrations of inosine, guanosine, xanthine, urate and neuron-specific enolase; and patients with tuberculous meningitis showed very high concentrations of inosine, xanthine and urate. Xanthine and urate concentrations were significantly higher in patients with tuberculous meningitis than in patients with viral or bacterial meningitis. These results suggest that in the acute stage of bacterial, viral and tuberculous meningitis, neuronal energy metabolism may be altered. The measurement of cerebrospinal xanthine and uric acid concentrations may be useful for the early diagnosis of a tuberculous origin.

Dampened hippocampal oscillations and enhanced spindle activity in an asymptomatic model of developmental cortical malformations.

Developmental cortical malformations comprise a large spectrum of histopathological brain abnormalities and syndromes. Their genetic, developmental and clinical complexity suggests they should be better understood in terms of the complementary action of independently timed perturbations (i.e., the multiple-hit hypothesis). However, understanding the underlying biological processes remains puzzling. Here we induced developmental cortical malformations in offspring, after intraventricular injection of methylazoxymethanol (MAM) in utero in mice. We combined extensive histological and electrophysiological studies to characterize the model. We found that MAM injections at E14 and E15 induced a range of cortical and hippocampal malformations resembling histological alterations of specific genetic mutations and transplacental mitotoxic agent injections. However, in contrast to most of these models, intraventricularly MAM-injected mice remained asymptomatic and showed no clear epilepsy-related phenotype as tested in long-term chronic recordings and with pharmacological manipulations. Instead, they exhibited a non-specific reduction of hippocampal-related brain oscillations (mostly in CA1); including theta, gamma and HFOs; and enhanced thalamocortical spindle activity during non-REM sleep. These data suggest that developmental cortical malformations do not necessarily correlate with epileptiform activity. We propose that the intraventricular in utero MAM approach exhibiting a range of rhythmopathies is a suitable model for multiple-hit studies of associated neurological disorders.

SU-8 based microprobes for simultaneous neural depth recording and drug delivery in the brain.

While novel influential concepts in neuroscience bring the focus to local activities generated within a few tens of cubic micrometers in the brain, we are still devoid of appropriate tools to record and manipulate pharmacologically neuronal activity at this fine scale. Here we designed, fabricated and encapsulated microprobes for simultaneous depth recording and drug delivery using exclusively the polymer SU-8 as structural material. A tetrode- and linear-like electrode patterning was combined for the first time with single and double fluidic microchannels for independent drug delivery. The device was tested experimentally using the in vivo anesthetized rat preparation. Both probe types successfully recorded detailed spatiotemporal features of local field potentials and single-cell activity at a resolution never attained before with integrated fluidic probes. Drug delivery was achieved with high spatial and temporal precision in a range from tens of nanoliters to a few microliters, as confirmed histologically. These technological advancements will foster a wide range of neural applications aimed at simultaneous monitoring of brain activity and delivery at a very precise micrometer scale.

Different emotional disturbances in two experimental models of temporal lobe epilepsy in rats.

Affective symptoms such as anxiety and depression are frequently observed in patients with epilepsy. The mechanisms of comorbidity of epilepsy and affective disorders, however, remain unclear. Diverse models are traditionally used in epilepsy research, including the status epilepticus (SE) model in rats, which are aimed at generating chronic epileptic animals; however, the implications of different SE models and rat strains in emotional behaviors has not been reported. To address this issue, we examined the emotional sequelae of two SE models of temporal lobe epilepsy (TLE)--the lithium-pilocarpine (LIP) model and the kainic acid (KA) model--in two different rat strains (Wistar and Sprague-Dawley), which differ significantly in the pattern and extent of TLE-associated brain lesions. We found differences between LIP- and KA-treated animals in tests for depression-like and anxiety-like behaviors, as well as differences in plasma corticosterone levels. Whereas only LIP-treated rats displayed increased motivation to consume saccharin, both SE models led to reduced motivation for social contact, with LIP-treated animals being particularly affected. Evaluation of behavior in the open field test indicated very low levels of anxiety in LIP-treated rats and a mild decrease in KA-treated rats compared to controls. After exposure to a battery of behavioral tests, plasma corticosterone levels were increased only in LIP-treated animals. This hyperactivity in the hypothalamus-pituitary-adrenocortical (HPA) axis was highly correlated with performance in the open field test and the social interaction test, suggesting that comorbidity of epilepsy and emotional behaviors might also be related to other factors such as HPA axis function. Our results indicate that altered emotional behaviors are not inherent to the epileptic condition in experimental TLE; instead, they likely reflect alterations in anxiety levels related to model-dependent dysregulation of the HPA axis.