P. M. Balaban

Retrograde signalling with nitric oxide at neocortical synapses.

Long‐term changes of synaptic transmission in slices of rat visual cortex were induced by intracellular tetanization: bursts of short depolarizing pulses applied through the intracellular electrode without concomitant presynaptic stimulation. Long‐term synaptic changes after this purely postsynaptic induction were associated with alterations of release indices, thus providing a case for retrograde signalling at neocortical synapses. Both long‐term potentiation and long‐term depression were accompanied by presynaptic changes, indicating that retrograde signalling can achieve both up‐ and down‐regulation of transmitter release. The direction and the magnitude of the amplitude changes induced by a prolonged intracellular tetanization depended on the initial properties of the input. The inputs with initially high paired‐pulse facilitation (PPF) ratio, indicative of low release probability, were most often potentiated. The inputs with initially low PPF ratio, indicative of high release probability, were usually depressed or did not change. Thus, prolonged postsynaptic activity can lead to normalization of the weights of nonactivated synapses. The dependence of polarity of synaptic modifications on initial PPF disappeared when plastic changes were induced with a shorter intracellular tetanization, or when the NO signalling pathway was interrupted by inhibition of NO synthase activity or by application of NO scavengers. This indicates that the NO‐dependent retrograde signalling system has a relatively high activation threshold. Long‐term synaptic modifications, induced by a weak postsynaptic challenge or under blockade of NO signalling, were nevertheless associated with presynaptic changes. This suggests the existence of another retrograde signalling system, additional to the high threshold, NO‐dependent system. Therefore, our data provide a clear case for retrograde signalling at neocortical synapses and indicate that multiple retrograde signalling systems, part of which are NO‐dependent, are involved.

POSTEMBRYONIC NEURONOGENESIS IN THE PROCEREBRUM OF THE TERRESTRIAL SNAIL, HELIX LUCORUM L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70-80%) of the snail procerebrum are produced during the first 1-2 months of posthatching development.

Neural mechanisms of age-dependent changes in avoidance behaviour of the snail Helix lucorum

In order to study the emergence of different components of learning and memory, we investigated developmental changes in behavioural, neurophysiological and histochemical experiments in newborn and adult terrestrial snails (Helix lucorum L.). The absence of sensitization and inability to develop avoidance-conditioned reactions were revealed in behavioral experiments in juvenile snails under 1 month old. Investigation of neural mechanisms of this behavioral deficit showed the absence of sensitization in spike reactions to repeated nerve stimulation in command neurons for the avoidance behaviour in juvenile snails. The same dynamics of response amplitude, as in juvenile snails, was seen in adults only after selective impairment of serotonergic neurons by treatment with 5,7-dihydroxytryptamine. During 1 month after hatching, the serotonin content investigated with fluorescence techniques was very low in the nervous system. This result suggests that the absence of sensitization, as well as inability to be aversively conditioned is related to 5-hydroxytryptamine level, which changes during postnatal development in the snail.

Neuronal correlates of aversive learning in command neurons for avoidance behavior ofHelix lucorum L.

Changes in responsiveness to food and noxious stimuli were studied in interneurons controlling feeding behavior and in putative command neurons for avoidance behavior after 10-15 paired presentations of food and electrical shock in the pulmonate snail Helix lucorum L. It was shown earlier that such aversive learning procedure is associative, and the behavioral aversion to the reinforced type of food in intact snails is maintained for at least 21 days, while normal feeding behavior can be evoked by another type of food. Responses to food presentation in the feeding behavior interneurons changed only in pattern after learning procedure, while in the command neurons for avoidance behavior a new spike reaction appeared, which is assumed to be responsible for the behavioral changes. A possible mechanism of a conditioned reaction in the command neurons for avoidance behavior is discussed.

Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH

Significance We identified a human-specific endogenous retroviral insert (hsERV) that acts as an enhancer for human PRODH, hsERV_PRODH. PRODH encodes proline dehydrogenase, which is involved in neuromediator synthesis in the CNS. We show that the hsERV_PRODH enhancer acts synergistically with the CpG island of PRODH and is regulated by methylation. We detected high PRODH expression in the hippocampus, which was correlated with the undermethylated state of this enhancer. PRODH regulatory elements provide neuron-specific transcription in hippocampal cells, and the mechanism of hsERV_PRODH enhancer activity involves the binding of transcriptional factor SOX2. Because PRODH is associated with several neurological disorders, we hypothesize that the human-specific regulation of PRODH by hsERV_PRODH may have played a role in human evolution by upregulating the expression of this important CNS-specific gene. Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERVPRODH, that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERVPRODH enhancer activity. We showed that the hsERVPRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERVPRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERVPRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERVPRODH and PRODH may have contributed to human CNS evolution.

Pedal serotonergic neurons modulate the synaptic input of withdrawal interneurons ofHelix

A group of serotonergic cells, located in the pedal ganglia ofHelix lucorum, modulates synaptic responses of neurons involved in withdrawal behavior. Extracellular or intracellular stimulation of these serotonergic cells leads to facilitation of spike responses to noxious stimuli in the putative command neurons for withdrawal behavior. Noxious tactile stimuli elicit an increase in background spiking frequency in the modulatory neurons and a corresponding increase in stimulus-evoked spike responses in withdrawal interneurons. The serotonergic neurons have processes in the neuropil of the parieto-visceral ganglia complex, consistent with their putative role in modulating the activity of giant parietal interneurons, which send processes to the same neuropil and to the pedal ganglia. The serotonergic cells respond to noxious tactile and chemical stimuli. Although the group as a whole respond to noxious stimuli applied to any part of the body, most cells respond more to ipsilateral than contralateral stimulation, and exhibit differences in receptive areas. Intracellular investigation revealed electrical coupling between serotonergic neurons which could underlie the recruitment of members of the group not responding to a given noxious stimulus.

A Single Serotonergic Modulatory Cell Can Mediate Reinforcement in the Withdrawal Network of the Terrestrial Snail

A cluster of 40 serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in the modulation of withdrawal behavior and to be necessary during the acquisition of aversive withdrawal conditioning in intact snails. Local extracellular stimulation of the serotonergic cells paired with a test stimulus elicited a pairing-specific increase (the difference between paired and explicitly unpaired sessions was significant, p <.01) of synaptic responses to test stimulation in the premotor interneurons involved in withdrawal. This result suggested participation of serotonergic cells in mediating the reinforcement in the withdrawal network. Intracellular stimulation of only one identified Pd4 cell from the pedal group of serotonergic neurons paired with a test stimulus also significantly increased (the difference between paired and explicitly unpaired sessions was significant, p <.05) synaptic responses to paired nerve stimulation in same premotor interneurons involved in withdrawal. Morphological investigation of a cluster of pedal serotonergic neurons showed that only the Pd4 cell had branches in the parietal ganglia neuropile where the synapses of premotor withdrawal interneurons and of presynaptic neurons are located. The data suggest that a single serotonergic cell can mediate the reinforcement in the withdrawal network of the terrestrial snail. Patterns of responses of the Pd4 cells to tactile and chemical stimuli conform to the suggestion.

The synapse between LE sensory neurons and gill motoneurons makes only a small contribution to the Aplysia gill-withdrawal reflex.

The monosynaptic connection between the mechano‐sensory neurons in the LE cluster and gill motoneurons has been extensively studied and used as a model for the gill‐withdrawal reflex and its behavioural plasticity. In an attempt to evaluate the contribution of this synapse to the behaviour, we used voltage‐sensitive dye recording to determine the number of activated LE neurons and the number of spikes made by each neuron in response to a light touch. In five preparations, light touch activated a median of five sensory cells with a median of 1.6 spikes per cell. From a comparison of the sizes of the motoneuron synaptic potentials elicited by LE spikes and elicited by a light siphon touch, we estimate that the LE sensory neurons contribute ˜5% of the motoneuron synaptic potential in response to this touch. This result casts doubt on the validity of using this synaptic connection as a model for gill‐withdrawal behaviour. Siphon nerve recordings reveal the existence of short‐latency, low‐threshold neurons that may provide much of the sensory input in response to a light touch.

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.

BACKGROUND SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. METHODS Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. RESULTS Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. CONCLUSIONS SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. GENERAL SIGNIFICANCE The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

Putative neuropeptides and an EF-hand motif region are encoded by a novel gene expressed in the four giant interneurons of the terrestrial snail.

Nine giant interneurons located in the pleural and parietal ganglia of the terrestrial snail Helix lucorum L. were reported to be a key element in the network controlling withdrawal behaviour of the animal. Using a combination of complementary DNA subtraction cloning and differential screening approaches we have isolated a novel gene named HCS2 which is expressed predominantly in a subset of these interneurons. The predicted amino acid sequence of the HCS2 protein contains at the N-terminus a hydrophobic leader sequence and four putative neuropeptides, and at the C-terminus a perfect match to the consensus motif of the EF-hand family of the Ca2+-binding proteins. All four predicted neuropeptides bear a C-terminal signature sequence Tyr-Pro-Arg-X (where X is Ile, Leu, Val or Pro), and three of them are likely to be amidated. Physiological action of three synthetic peptides corresponding to the predicted mature HCS2 peptides mimics fairly well the described action of parietal interneurons on follower motoneurons controlling pneumostome closure. In situ hybridization experiments demonstrated that the HCS2 gene is selectively expressed in the four parietal giant interneurons, as well as in several small unidentified neurons. The onset of the HCS2 transcription during embryogenesis coincides temporally with the time-point when the first withdrawal responses of the embryo to tactile stimulation appear. We propose that the HCS2 gene encodes a hybrid precursor protein whose processed products act as neuromodulators or neurotransmitters mediating the withdrawal reactions of the snail, and in addition may participate in the calcium regulatory pathways or calcium homeostasis in command neurons.