Cerebellar cortex involvement in classical eyelid conditioning

A model for cerebellar involvement in motor learning was tested using classical eyelid conditioning in the rabbit. Briefly, we assume that modifications of the strength of granule cell synapses at Purkinje cells in the cerebellar cortex and mossy fiber (MF) synapses at cerebellar interpositus nuclei are responsible for the acquisition, adaptively-timed expression, and extinction of conditioned eyelid responses (CRs). A corollary of these assumptions is that the cerebellar cortex is necessary for acquisition and extinction. This model also suggests a mechanism whereby the cerebellar cortex can discriminate different times during a conditioned stimulus (CS) and thus mediate the learned timing of CRs. Therefore, experiments were done to determine the role of the cerebellar cortex in the timing, extinction, and acquisition of CRs. Lesions of the cerebellar cortex that included the anterior lobe disrupted the learned timing of CRs such that they occurred at extremely short latencies. Stimulation of MFs in the middle cerebellar peduncle as the CS could support differently timed CRs in the same animal. These data indicate that synaptic plasticity in the cerebellar cortex mediates the learned timing of CRs. These short-latency CRs which resulted from anterior lobe damage did not extinguish, while CRs in animals receiving lesions which did not include the anterior lobe extinguished normally. Preliminary data suggests that lesions of the anterior lobe which produce short-latency responses prevent the acquisition of CRs to a novel CS. These findings indicate that the anterior lobe of cerebellar cortex is necessary for eyelid conditioning. The involvement of the anterior lobe in eyelid conditioning has not been previously reported, however, the anterior lobe has generally been spared in lesion studies examining cerebellar cortex involvement in eyelid conditioning due to its relatively inaccessible location. The observation that the anterior lobe of the cerebellar cortex is not always required for the basic expression of CRs, but is necessary for response timing, extinction, and acquisition, is consistent with the hypothesis that eyelid conditioning can involve plasticity in both the cerebellar cortex and interpositus nucleus and that plasticity in the nucleus is controlled by Purkinje cell activity. ^

Glutamate iontophoresis induces long-term potentiation in the absence of evoked presynaptic activity

$\rm\underline{L}$ong-$\rm\underline{t}$erm $\rm\underline{p}$otentiation (LTP) is a candidate cellular mechanism underlying mammalian learning and memory. Protocols that induce LTP typically involve afferent stimulation. The experiments described in this dissertation tested the hypothesis that LTP induction does not require presynaptic activity. The significance of this hypothesis is underscored by results suggesting that LTP expression may involve activity-dependent presynaptic changes. An induction protocol using glutamate iontophoresis was developed that reliably induces LTP in hippocampal slices without afferent stimulation (ionto-LTP). Ionto-LTP is induced when excitatory postsynaptic potentials are completely blocked with adenosine and $\rm\underline{t}$etrodo$\rm\underline{t}$o$\rm\underline{x}$in (TTX). These results suggest constraints on the involvement of presynaptic mechanisms and putative retrograde messengers in LTP induction and expression; namely, these processes must function without many forms of activity-dependent presynaptic processes.^ In testing the role of pre-and postsynaptic mechanisms in LTP expression whole-cell recordings were used to examine the frequency and amplitude of $\rm\underline{s}$pontaneous $\rm\underline{e}$xcitatory $\rm\underline{p}$o$\rm\underline{s}$ynaptic $\rm\underline{c}$urrents (sEPSCs) in CA1 pyramidal neurons. sEPSCs where comprised of an equal mixture of TTX insensitive miniature EPSCs and sEPSCs that appeared to result from spontaneous action potentials (i.e., TTX sensitive EPSCs). The detection of all sEPSCs was virtually eliminated by CNQX, suggesting that sEPSCs were glutamate mediated synaptic events. Changes in the amplitude and frequency sEPSCs were examined during the expression of ionto-LTP to obtain new information about the cellular location of mechanisms involved in synaptic plasticity. The findings of this dissertation show that ionto-LTP expression results from increased sEPSC amplitude in the absence of lasting increases in sEPSC frequency. Potentiation of sEPSC amplitude without changes in sEPSC frequency has been previously interpreted to be due to postsynaptic mechanisms. Although this interpretation is supported by findings from peripheral synapses, its application to the central nervous system is unclear. Therefore, alternative mechanisms are also considered in this dissertation. Models based on increased release probability for action potential dependent transmitter release appear insufficient to explain our results. The most straightforward interpretation of the results in this dissertation is that LTP induced by glutamate iontophoresis on dendrites of CA1 pyramidal neurons is mediated by postsynaptic mechanisms. ^

Notch signaling in the brain: in good and bad times.

Notch signaling is an evolutionarily conserved pathway, which is fundamental for neuronal development and specification. In the last decade, increasing evidence has pointed out an important role of this pathway beyond embryonic development, indicating that Notch also displays a critical function in the mature brain of vertebrates and invertebrates. This pathway appears to be involved in neural progenitor regulation, neuronal connectivity, synaptic plasticity and learning/memory. In addition, Notch appears to be aberrantly regulated in neurodegenerative diseases, including Alzheimer's disease and ischemic injury. The molecular mechanisms by which Notch displays these functions in the mature brain are not fully understood, but are currently the subject of intense research. In this review, we will discuss old and novel Notch targets and molecular mediators that contribute to Notch function in the mature brain and will summarize recent findings that explore the two facets of Notch signaling in brain physiology and pathology.

Topographic sleep EEG changes in the acute and chronic stage of hemispheric stroke.

After stroke, the injured brain undergoes extensive reorganization and reconnection. Sleep may play a role in synaptic plasticity underlying stroke recovery. To test this hypothesis, we investigated topographic sleep electroencephalographic characteristics, as a measure of brain reorganization, in the acute and chronic stages after hemispheric stroke. We studied eight patients with unilateral stroke in the supply territory of the middle cerebral artery and eight matched controls. All subjects underwent a detailed clinical examination including assessment of stroke severity, sleep habits and disturbances, anxiety and depression, and high-density electroencephalogram examination with 128 electrodes during sleep. The recordings were performed within 10 days after stroke in all patients, and in six patients also 3 months later. During sleep, we found higher slow-wave and theta activity over the affected hemisphere in the infarct area in the acute and chronic stage of stroke. Slow-wave, theta activity and spindle frequency range power over the affected hemisphere were lower in comparison to the non-affected side in a peri-infarct area in the patients' group, which persisted over time. Conversely, in wakefulness, only an increase of delta, theta activity and a slowing of alpha activity over the infarct area were found. Sleep slow-wave activity correlated with stroke severity and outcome. Stroke might have differential effects on the generation of delta activity in wakefulness and sleep slow waves (1-8 Hz). Sleep electroencephalogram changes over both the affected and non-affected hemispheres reflect the acute dysfunction caused by stroke and the plastic changes underlying its recovery. Moreover, these changes correlate with stroke severity and outcome.

Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

Identification of a new genomic hot spot of evolutionary diversification of protein function.

Establishment of phylogenetic relationships remains a challenging task because it is based on computational analysis of genomic hot spots that display species-specific sequence variations. Here, we identify a species-specific thymine-to-guanine sequence variation in the Glrb gene which gives rise to species-specific splice donor sites in the Glrb genes of mouse and bushbaby. The resulting splice insert in the receptor for the inhibitory neurotransmitter glycine (GlyR) conveys synaptic receptor clustering and specific association with a particular synaptic plasticity-related splice variant of the postsynaptic scaffold protein gephyrin. This study identifies a new genomic hot spot which contributes to phylogenetic diversification of protein function and advances our understanding of phylogenetic relationships.

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

Unique regulatory properties of the type 2a Ca2+ channel β subunit caused by palmitoylation

β subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for β2a, which differs in that it does not support prepulse facilitation of α1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal α1E Ca2+ channels, and is more effective in blocking inhibition of α1E channels by G protein-coupled receptors. We show that the distinguishing properties of β2a, rather than interaction with a distinct site of α1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of β2a were lost in a mutant that could not be palmitoylated [β2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three β2 splice variants differing only in their N-termini.

All-or-none potentiation at CA3-CA1 synapses

The molecular mechanisms underlying long-term potentiation in the hippocampus have received much attention because of the likely functional importance of synaptic plasticity for information storage and the development of neuronal connectivity. Surprisingly, it remains unclear whether activity modifies the strength of individual synapses in a digital (all-or-none) or analog (graded) manner. Here we characterize step-like all-or-none transitions from baseline synaptic transmission to potentiated states following protocols for inducing potentiation at putative single CA3-CA1 synaptic connections. Individual synapses appear to have all-or-none potentiation indicative of highly cooperative processes but different thresholds for undergoing potentiation. These results raise the possibility that some forms of synaptic memory may be stored in a digital manner in the brain.

A neuromodulatory role of interleukin-1β in the hippocampus

It is widely accepted that interleukin-1β (IL-1β), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1β gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-d-aspartate (NMDA) receptor antagonist, (±)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1β in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.

Bidirectional, experience-dependent regulation of N-methyl-d-aspartate receptor subunit composition in the rat visual cortex during postnatal development

In the visual cortex, as elsewhere, N-methyl-d-aspartate receptors (NMDARs) play a critical role in triggering long-term, experience-dependent synaptic plasticity. Modifications of NMDAR subunit composition alter receptor function, and could have a large impact on the properties of synaptic plasticity. We have used immunoblot analysis to investigate the effects of age and visual experience on the expression of different NMDAR subunits in synaptoneurosomes prepared from rat visual cortices. NMDARs at birth are comprised of NR2B and NR1 subunits, and, over the first 5 postnatal weeks, there is a progressive inclusion of the NR2A subunit. Dark rearing from birth attenuates the developmental increase in NR2A. Levels of NR2A increase rapidly (in <2 hr) when dark-reared animals are exposed to light, and decrease gradually over the course of 3 to 4 days when animals are deprived of light. These data reveal that NMDAR subunit composition in the visual cortex is remarkably dynamic and bidirectionally regulated by sensory experience. We propose that NMDAR subunit regulation is a mechanism for experience-dependent modulation of synaptic plasticity in the visual cortex, and serves to maintain synaptic strength within an optimal dynamic range.

Experience-dependent, asymmetric expansion of hippocampal place fields

Theories of sequence learning based on temporally asymmetric, Hebbian long-term potentiation predict that during route learning the spatial firing distributions of hippocampal neurons should enlarge in a direction opposite to the animal’s movement. On a route AB, increased synaptic drive from cells representing A would cause cells representing B to fire earlier and more robustly. These effects appeared within a few laps in rats running on closed tracks. This provides indirect evidence for Hebbian synaptic plasticity and a functional explanation for why place cells become directionally selective during route following, namely, to preserve the synaptic asymmetry necessary to encode the sequence direction.

Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.

Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice

Calretinin (Cr) is a Ca2+ binding protein present in various populations of neurons distributed in the central and peripheral nervous systems. We have generated Cr-deficient (Cr−/−) mice by gene targeting and have investigated the associated phenotype. Cr−/− mice were viable, and a large number of morphological, biochemical, and behavioral parameters were found unaffected. In the normal mouse hippocampus, Cr is expressed in a widely distributed subset of GABAergic interneurons and in hilar mossy cells of the dentate gyrus. Because both types of cells are part of local pathways innervating dentate granule cells and/or pyramidal neurons, we have explored in Cr−/− mice the synaptic transmission between the perforant pathway and granule cells and at the Schaffer commissural input to CA1 pyramidal neurons. Cr−/− mice showed no alteration in basal synaptic transmission, but long-term potentiation (LTP) was impaired in the dentate gyrus. Normal LTP could be restored in the presence of the GABAA receptor antagonist bicuculline, suggesting that in Cr−/− dentate gyrus an excess of γ-aminobutyric acid (GABA) release interferes with LTP induction. Synaptic transmission and LTP were normal in CA1 area, which contains only few Cr-positive GABAergic interneurons. Cr−/− mice performed normally in spatial memory task. These results suggest that expression of Cr contributes to the control of synaptic plasticity in mouse dentate gyrus by indirectly regulating the activity of GABAergic interneurons, and that Cr−/− mice represent a useful tool to understand the role of dentate LTP in learning and memory.