Nav1.6 but not KCa3.1 channels contribute to heterogeneity in coding abilities and dynamics of action potentials in the L5 neocortical pyramidal neurons.

Electrophysiological and genetic studies reveal two major subclasses of layer 5 (L5) neocortical pyramidal neurons that differ in electrical parameters and afterhyperpolarization. KCa3.1 channels are identified as contributors to slow afterhyperpolarization (sAHP), and they are expressed by one subclass of L5 neurons. Yet, the impact of class-specific sAHP and KCa3.1 channels on coding abilities of the L5 neurons and dynamics of their action potentials (APs) remains poorly understood. Here, by comparing sAHP+ neurons to those with weak sAHP we investigate differences between the two groups in coding and AP features to address the question of whether those differences are due to contribution of KCa3.1 or other channels. Using patch clamp electrophysiology, channel blockers, and immunohistochemistry we demonstrate that Nav1.6 channels but not KCa3.1 channels affect the threshold of AP, its dynamics and coding abilities of the L5 cells. Immunohistochemical data show that KCa3.1+ and KCa3.1- neurons share the same pattern of Nav1.6 expression in the soma and axonal initial segment, thus they may differ in quantity of the channels expressed. Our study links the Nav1.6 function underlying regulation of voltage threshold to the abilities of L5 neurons to encode high frequencies.

Immediate-Early Genes Detection in the CNS of Terrestrial Snail.

In the present work, using in situ hybridization, we studied the expression patterns of three molluscan homologs of vertebrate immediate-early genes C/EBP, c-Fos, and c-Jun in the central nervous system (CNS) of terrestrial gastropod snail Helix. The molluscan C/EBP gene was described in literature, while c-Fos and c-Jun were studied in terrestrial snails for the first time. Localization of the expression was traced in normal conditions, and in preparations physiologically activated using stimulation of suboesophageal ganglia nerves. No expression was detected constitutively. In stimulated preparations, all three genes had individual expression patterns in Helix CNS, and the level of expression was stimulus-dependent. The number of cells expressing the gene of interest was different from the number of cells projecting to the stimulated nerve, and thus activated retrogradely. This difference depended on the ganglia studied. At the subcellular level, the labeled RNA was observed as dots (probably small clusters of RNA molecules) and shapeless mass of RNA, often seen as a circle at the internal border of the cell nuclei. The data provide a basis for further study of behavioral role of these putative immediate-early genes in snail behavior and learning.

Identification of Immediate Early Genes in the Nervous System of Snail Helix lucorum.

Immediate early genes (IEGs) are useful markers of neuronal activation and essential components of neuronal response. While studies of gastropods have provided many insights into the basic learning and memory mechanisms, the genome-wide assessment of IEGs has been mainly restricted to vertebrates. In this study, we identified IEGs in the terrestrial snail Helix lucorum In the absence of the genome, we conducted de novo transcriptome assembly using reads with short and intermediate lengths cumulatively covering more than 98 billion nucleotides. Based on this assembly, we identified 37 proteins corresponding to contigs differentially expressed (DE) in either the parietal ganglia (PaG) or two giant interneurons located within the PaG of the snail in response to the neuronal stimulation. These proteins included homologues of well-known mammalian IEGs, such as c-jun/jund, C/EBP, c-fos/fosl2, and Egr1, as well as homologues of genes not yet implicated in the neuronal response.

A BK channel-mediated feedback pathway links single-synapse activity with action potential sharpening in repetitive firing.

Action potential shape is a major determinant of synaptic transmission, and mechanisms of spike tuning are therefore of key functional significance. We demonstrate that synaptic activity itself modulates future spikes in the same neuron via a rapid feedback pathway. Using Ca2+ imaging and targeted uncaging approaches in layer 5 neocortical pyramidal neurons, we show that the single spike-evoked Ca2+ rise occurring in one proximal bouton or first node of Ranvier drives a significant sharpening of subsequent action potentials recorded at the soma. This form of intrinsic modulation, mediated by the activation of large-conductance Ca2+/voltage-dependent K+ channels (BK channels), acts to maintain high-frequency firing and limit runaway spike broadening during repetitive firing, preventing an otherwise significant escalation of synaptic transmission. Our findings identify a novel short-term presynaptic plasticity mechanism that uses the activity history of a bouton or adjacent axonal site to dynamically tune ongoing signaling properties.

Long-living RNA in the CNS of terrestrial snail.

Click-iT method can be used to trace the neurons where the newly synthesized RNA transcripts occur. Our experiments performed with the CNS of terrestrial mollusk Helix demonstrate that 5-ethynyluridine (EU) is selectively incorporated in RNA but not in DNA. The time of EU accumulation necessary for its detection was about several hours. EU was injected into the body cavity of adult mollusks, and was detectable in neurons for several days. In juveniles, EU was introduced via bathing of snails in the EU-containing saline, and was reliably detected within time period of several weeks. Our data suggest that short-living forms of RNA cannot be detected by Click-iT method, while the long-living forms of RNA can be spatially detected in individual neurons.

Adaptive Changes in the Vestibular System of Land Snail to a 30-Day Spaceflight and Readaptation on Return to Earth.

The vestibular system receives a permanent influence from gravity and reflexively controls equilibrium. If we assume gravity has remained constant during the species' evolution, will its sensory system adapt to abrupt loss of that force? We address this question in the land snail Helix lucorum exposed to 30 days of near weightlessness aboard the Bion-M1 satellite, and studied geotactic behavior of postflight snails, differential gene expressions in statocyst transcriptome, and electrophysiological responses of mechanoreceptors to applied tilts. Each approach revealed plastic changes in the snail's vestibular system assumed in response to spaceflight. Absence of light during the mission also affected statocyst physiology, as revealed by comparison to dark-conditioned control groups. Readaptation to normal tilt responses occurred at ~20 h following return to Earth. Despite the permanence of gravity, the snail responded in a compensatory manner to its loss and readapted once gravity was restored.

Encoding of High Frequencies Improves with Maturation of Action Potential Generation in Cultured Neocortical Neurons.

The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials (APs). Experimental results support this idea, however mechanisms of fast onset of APs in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of AP generation and encoding. Here we characterize properties of AP encoding in neocortical neurons grown for 11-25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and AP generation. The onset of APs becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of APs as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of AP generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of AP generation and encoding, and their maturation.

Impairment of the serotonergic neurons underlying reinforcement elicits extinction of the repeatedly reactivated context memory.

We analyzed changes in the activity of individually identifiable neurons involved in the networks underlying feeding and withdrawal behaviors in snails before, during, and after aversive learning in vitro. Responses to food in the "reinforcing" serotonergic neurons involved in withdrawal changed significantly after training, implying that these serotonergic cells participate in the reactivation of memory and are involved in the reconsolidation process. In behavioral experiments it was shown that impairment of the functioning of the serotonergic system with the selective neurotoxin 5,7-DiHT did not change the memory, when tested once, but resulted in a complete extinction of the contextual memory after repeated reactivation of memory. Conversely, the cued memory to a specific type of food was significantly reduced but still present. Thus, we conclude that it is only for the context memory, that participation of the "reinforcing" serotonergic neurons in memory retrieval may be the gate condition for the choice between extinction/reconsolidation.

RNA synthesis and turnover in the molluscan nervous system studied by Click-iT method.

RNA synthesis can be detected by means of the in vivo incorporation of 5-ethynyluridine (EU) in newly-synthesized RNA with the relatively simple Click-iT method. We used this method to study the RNA synthesis in the CNS tissue of adult and juvenile terrestrial snails Helix lucorum L. Temporally, first labeled neurons were detected in the adult CNS after 4-h of isolated CNS incubation in EU solution, while 12-h of incubation led to extensive labeling of most CNS neurons. The EU labeling was present as the nuclear and nucleolar staining. The cytoplasm staining was observed after 2-3 days of CNS washout following the EU exposure for 16 h. In juvenile CNS, the first staining reaction was apparent as the staining of apical region in the procerebral lobe of cerebral ganglia after 1h of CNS incubation in EU, while the maximum pattern of staining was obtained after 4h of CNS incubation. Thus, age-related differences in RNA synthesis are present. Activation of neurons elicited by serotonin and caffeine applications noticeably increased the intensity of staining. EU readily penetrates into the bodies of juvenile snails immersed in the EU solution. When the intact juvenile animals were immersed in the EU solution for 1h, the procerebrum staining, similar to the one detected in the incubated juvenile CNS, was observed.

Homolog of protein kinase Mζ maintains context aversive memory and underlying long-term facilitation in terrestrial snail Helix.

It has been shown that a variety of long-term memories in different regions of the brain and in different species are quickly erased by local inhibition of protein kinase Mζ (PKMζ), a persistently active protein kinase. Using antibodies to mammalian PKMζ, we describe in the present study the localization of immunoreactive molecules in the nervous system of the terrestrial snail Helix lucorum. Presence of a homolog of PKMζ was confirmed with transcriptomics. We have demonstrated in behavioral experiments that contextual fear memory disappeared under a blockade of PKMζ with a selective peptide blocker of PKMζ zeta inhibitory peptide (ZIP), but not with scrambled ZIP. If ZIP was combined with a "reminder" (20 min in noxious context), no impairment of the long-term contextual memory was observed. In electrophysiological experiments we investigated whether PKMζ takes part in the maintenance of long-term facilitation (LTF) in the neural circuit mediating tentacle withdrawal. LTF of excitatory synaptic inputs to premotor interneurons was induced by high-frequency nerve stimulation combined with serotonin bath applications and lasted at least 4 h. We found that bath application of 2 × 10(-6) M ZIP at the 90th min after the tetanization reduced the EPSP amplitude to the non-tetanized EPSP values. Applications of the scrambled ZIP peptide at a similar time and concentration didn't affect the EPSP amplitudes. In order to test whether effects of ZIP are specific to the synapses, we performed experiments with LTF of somatic membrane responses to local glutamate applications. It was shown earlier that serotonin application in such an "artificial synapse" condition elicits LTF of responses to glutamate. It was found that ZIP had no effect on LTF in these conditions, which may be explained by the very low concentration of PKMζ molecules in somata of these identified neurons, as evidenced by immunochemistry. Obtained results suggest that the Helix homolog of PKMζ might be involved in post-induction maintenance of long-term changes in the nervous system of the terrestrial snail.

Type 1 metalloproteinase is selectively expressed in adult rat brain and can be rapidly up-regulated by kainate.

The expression of metalloproteinase MMP-1 was traced in frontal sections of the rat brain in normal conditions and 4 h after an intraperitoneal injection of kainate. In the olfactory lobe, immunoreactivity was normally detected in the lateral olfactory tract. Kainate treatment led to the appearance of additional immunoreactivity in the neuropilar tracts. In the hippocampal part of brain, immunoreactive neurons were found exclusively after the kainate treatment in several hypothalamic and amygdalar nuclei, and in the restricted cortex areas (clusters of neurons in layers 3-4 of cortex, and a stripe of cells in layer 6). In the area between the hippocampus and cerebellum, MMP-1-like immunoreactivity was normally present in the entorhinal cortex, in the lateral periaqueductal gray, and in the pontine nucleus. After kainate treatment, the immunoreactive neurons were also found in the medial entorhinal cortex and in the dorsal raphe nucleus. In the brain stem, the immunoreactive cells were normally found in six nuclei. After kainate treatment, additional immunoreactivity appeared in the inferior olive neurons and in tracts supplying the cerebellar cortex. Thus, MMP-1 is present in several brain areas in normal conditions at a detectable level, and its expression increases after kainate-induced seizures.

Expression of the type 1 metalloproteinase in the rat hippocampus after the intracerebroventricular injection of ß-amyloid peptide (25-35).

The expression of matrix metalloproteinase of the first type was studied in frontal sections of the adult rat brain one month after a single intracerebroventricular injection of beta-amyloid peptide (25-35), which is known to be a well-known model of the development of Alzheimer's disease. Brain sections were stained immunocytochemically to detect MMP-1 expression, and histologically to reveal the state of hippocampal neurons. Administration of beta-amyloid peptide induced a significant degeneration of cells in the dorsal hippocampus. This was demonstrated by a significant decrease in the total number of cells and by the appearance of acidophilic neurons of altered (often triangular) shape. Altered cells were most often found in the hippocampal field CA3, and in a smaller quantity in the CA1 field. MMP-1-like immunoreactivity was found in the same hippocampal areas, the staining being restricted to the cells of altered shape (staining of somata and primary neurites). The data suggest possible involvement of the type 1 metalloproteinase in the development of Alzheimer's disease.

Biolistic delivery of voltage-sensitive dyes for fast recording of membrane potential changes in individual neurons in rat brain slices.

Optical recording of membrane potential changes with fast voltage-sensitive dyes (VSDs) in neurons is one of the very few available methods for studying the generation and propagation of electrical signals to the distant compartments of excitable cells. The more lipophilic is the VSD, the better signal-to-noise ratio of the optical signal can be achieved. At present there are no effective ways to deliver water-insoluble dyes into the membranes of live cells. Here, we report a possibility to stain individual live neurons with highly lipophilic VSDs in acute brain slices using biolistic delivery. We tested four ANEP-based VSDs with different lipophilic properties and showed their ability to stain single neurons in a slice area of up to 150 µm in diameter after being delivered by a biolistic apparatus. In the slices of neocortex and hippocampus, the two most lipophilic dyes, di-8-ANEPPS and di-12-ANEPPQ, showed cell-specific loading and Golgi-like staining patterns with minimal background fluorescence. Simultaneous patch-clamp and optical recording of biolistically stained neurons demonstrated a good match of optical and electrical signals both for spontaneous APs (action potentials) and stimulus-evoked events. Our results demonstrate the high efficiency of a fast and targeted method of biolistic delivery of lipophilic VSDs for optical signals recording from mammalian neurons in vitro.

Functional changes in the snail statocyst system elicited by microgravity.

BACKGROUND: The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. METHODOLOGY/PRINCIPAL FINDINGS: In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13-42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. CONCLUSIONS/SIGNIFICANCE: Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system's response to conditions on Earth and in space.

Family of CNP neuropeptides: common morphology in various invertebrates.

Neuropeptides expressed in the command neurons for withdrawal behavior were originally detected in the the central nervous system (CNS) of the terrestrial snail Helix (command neurons peptides, CNP). The family of CNP-like neuropeptides bears a C-terminal signature sequence Tyr-Pro-Arg-X. Using antisera against two of them, we have studied the CNS of various invertebrates belonging to the phyla of mollusks, annelids and insects. The immunoreactive neurons were detected in all studied species. Stained neurons were either interneurons projecting along the CNS ganglia chain, or sensory neurons, or neurohormonal cells. Beyond common morphological features, the immunoreactive cells had another similarity: the level of CNP expression depended on the functional state of the animal. Thus, the homologous neuropeptides in evolutionary distant invertebrate species possess some common morphological and functional features.

Two morphological sub-systems within the olfactory organs of a terrestrial snail.

In the present work, we have re-visited the problem of the olfactory neural system organization in the terrestrial snail. By staining the tentacle's nerves and their intrinsic tracts in different points of the cerebral ganglia-tentacles system we have found that the relatively small part of the primary sensory neurons from the sensory pad (7-8%) send their axons directly to the cerebral ganglia. The axons terminated in the metacerebral neuropil which suggests these receptors being not chemosensory but rather mechanosensory neurons. Majority of the primary sensory neurons are synaptically switching in the areas outside the cerebral ganglia, i.e. digits, glomeruli, tentacular ganglion. No primary sensory neurons of the olfactory pad were projecting directly to the procerebrum - the putative centre of olfactory information processing. The afferent tract innervating the procerebrum neuropil originated from the interneurons located in the tentacle ganglion and its digits. Our results suggest the presence of two different sub-systems within the snail nose - mechanosensory and chemosensory - with two different projection targets.

Primary sensory neurons containing command neuron peptide constitute a morphologically distinct class of sensory neurons in the terrestrial snail.

In the central nervous system of the terrestrial snail Helix, the gene HCS2, which encodes several neuropeptides of the CNP (command neuron peptide) family, is mostly expressed in cells related to withdrawal behavior. In the present work, we demonstrate that a small percentage (0.1%) of the sensory cells, located in the sensory pad and in the surrounding epithelial region ("collar") of the anterior and posterior tentacles, is immunoreactive to antisera raised against the neuropeptides CNP2 and CNP4, encoded by the HCS2 gene. No CNP-like-immunoreactive neurons have been detected among the tentacular ganglionic interneurons. The CNP-like-immunoreactive fiber bundles enter the cerebral ganglia within the nerves of the tentacles (tentacular nerve and medial lip nerve) and innervate the metacerebral lobe, viz., the integrative brain region well-known as the target area for many cerebral ganglia nerves. The procerebral lobe, which is involved in the processing of olfactory information, is not CNP-immunoreactive. Our data suggest that the sensory cells, which contain the CNP neuropeptides, belong to a class of sensory neurons with a specific function, presumably involved in the withdrawal behavior of the snail.

Neuropeptides of Drosophila related to molluscan neuropeptides: dependence of the immunoreactivity pattern on the ontogenetic stage and functional state.

The CNP neuropeptides (Command Neuron Peptides) were first found in the command neurons for withdrawal behavior in the terrestrial snail. Given the fact that certain peptides can be found in various invertebrates, we examined Drosophila brains to determine if CNP-like peptides were present. Two types of antisera were used: one against CNP2, which was expected to recognize peptide products of the genes "hugin", "capa", CG6371, and a second antiserum against CNP4, which was expected to recognize neuropeptides encoded by the gene "capa" only. In larvae, both antibodies stained the abdominal perisympathetic organ, and several groups of neurons in the suboesophageal ganglia, while two neuronal clusters in the protocerebrum were stained with CNP2 antibody exclusively. The set of peptidergic neurons was conserved throughout all larval development. In the suboesophageal ganglia, the number of immunoreactive neurons apparently decreased at the pupae stage, and only one pair of large neurons in the caudal part of the suboesophageal ganglia was detected in adults. Experimental body injury led in the adult fruit flies to appearance of additional immunoreactive neurons, the pattern of staining becoming similar to that in larval CNS. The study demonstrates that the number of neurons expressing CNP-like immunoreactivity depends on the developmental stage and functional state of the animal, and that the CNP2-like and CNP4-like neuropeptides are colocalized in some neurons. We conclude that the family of CNP-like neuropeptides seems to be common for various invertebrate phyla.

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.