Oxygen/Glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection

Differential aberrant sprouting in temporal lobe epilepsy with psychiatric co-morbidities

Psychiatric co-morbidities in epilepsy are common in patients with temporal lobe epilepsy (TLE). Pathological alterations in TLE are well characterised; however, neuropathologic data are relatively scale regarding the association between psychiatric diseases and epilepsy. Our objective was to evaluate the clinical data of 46 adult TLE patients with and without psychiatric co-morbidities and to correlate the data with hippocampal neuronal density and mossy fiber sprouting. Accordingly, patients were grouped as follows: TLE patients without history of psychiatric disorder (TLE, n = 16), TLE patients with interictal psychosis (TLE + P, n = 14), and TLE patients with major depression (TLE + D, n = 16). Hippocampi from autopsies served as non-epileptic controls (n = 10). TLE + P exhibited significantly diminished mossy fiber sprouting and decreased neuronal density in the entorhinal cortex when compared with TLE. TLE + P showed significantly poorer results in verbal memory tasks. TLE + D exhibited significantly increased mossy fiber sprouting length when compared with TLE and TLE + P. Further, a higher proportion of TLE + D and TLE + P presented secondarily generalised seizures than did TLE. Our results indicate that TLE patients with psychiatric disorders have distinct features when compared with TLE patients without psychiatric co-morbidities and that these changes may be involved in either the manifestation or the maintenance of psychiatric co-morbidities in epilepsy. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

New ways of looking at synapses

Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.

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. ^

Cerebellar mechanisms for motor learning: Testing predictions from a large-scale computer simulation

The cerebellum is the major brain structure that contributes to our ability to improve movements through learning and experience. We have combined computer simulations with behavioral and lesion studies to investigate how modification of synaptic strength at two different sites within the cerebellum contributes to a simple form of motor learning—Pavlovian conditioning of the eyelid response. These studies are based on the wealth of knowledge about the intrinsic circuitry and physiology of the cerebellum and the straightforward manner in which this circuitry is engaged during eyelid conditioning. Thus, our simulations are constrained by the well-characterized synaptic organization of the cerebellum and further, the activity of cerebellar inputs during simulated eyelid conditioning is based on existing recording data. These simulations have allowed us to make two important predictions regarding the mechanisms underlying cerebellar function, which we have tested and confirmed with behavioral studies. The first prediction describes the mechanisms by which one of the sites of synaptic modification, the granule to Purkinje cell synapses (gr → Pkj) of the cerebellar cortex, could generate two time-dependent properties of eyelid conditioning—response timing and the ISI function. An empirical test of this prediction using small, electrolytic lesions of the cerebellar cortex revealed the pattern of results predicted by the simulations. The second prediction made by the simulations is that modification of synaptic strength at the other site of plasticity, the mossy fiber to deep nuclei synapses (mf → nuc), is under the control of Purkinje cell activity. The analysis predicts that this property should confer mf → nuc synapses with resistance to extinction. Thus, while extinction processes erase plasticity at the first site, residual plasticity at mf → nuc synapses remains. The residual plasticity at the mf → nuc site confers the cerebellum with the capability for rapid relearning long after the learned behavior has been extinguished. We confirmed this prediction using a lesion technique that reversibly disconnected the cerebellar cortex at various stages during extinction and reacquisition of eyelid responses. The results of these studies represent significant progress toward a complete understanding of how the cerebellum contributes to motor learning. ^

Chronic stress alters synaptic terminal structure in hippocampus

Repeated psychosocial or restraint stress causes atrophy of apical dendrites in CA3 pyramidal neurons of the hippocampus, accompanied by specific cognitive deficits in spatial learning and memory. Excitatory amino acids mediate this atrophy together with adrenal steroids and the neurotransmitter serotonin. Because the mossy fibers from dentate granule neurons provide a major excitatory input to the CA3 proximal apical dendrites, we measured ultrastructural parameters associated with the mossy fiber–CA3 synapses in control and 21-day restraint-stressed rats in an effort to find additional morphological consequences of stress that could help elucidate the underlying anatomical as well as cellular and molecular mechanisms. Although mossy fiber terminals of control rats were packed with small, clear synaptic vesicles, terminals from stressed animals showed a marked rearrangement of vesicles, with more densely packed clusters localized in the vicinity of active zones. Moreover, compared with controls, restraint stress increased the area of the mossy fiber terminal occupied by mitochondrial profiles and consequently, a larger, localized energy-generating capacity. A single stress session did not produce these changes either immediately after or the next day following the restraint session. These findings provide a morphological marker of the effects of chronic stress on the hippocampus that points to possible underlying neuroanatomical as well as cellular and molecular mechanisms for the ability of repeated stress to cause structural changes within the hippocampus.

Microglia in the cerebral and cerebellar cortices in individuals with autism

In this thesis, we explore the density of the microglia in the cerebral and cerebellar cortices of individuals with autism to investigate the hypothesis that neuroinflammation is involved in autism. We describe in our findings an increase in microglial density in two disparate cortical regions, frontal insular cortex and visual cortex, in individuals with autism (Tetreault et al., 2012). Our results imply that there is a global increase in the microglial density and neuroinflammation in the cerebral cortex of individuals with autism.

We expanded our cerebellar study to additional neurodevelopmental disorders that exhibit similar behaviors to autism spectrum disorder and have known cerebellar pathology. We subsequently found a more than threefold increase in the microglial density specific to the molecular layer of the cerebellum, which is the region of the Purkinje and parallel fiber synapses, in individuals with autism and Rett syndrome. Moreover, we report that not only is there an increase in microglia density in the molecular layer, the microglial cell bodies are significantly larger in perimeter and area in individuals with autism spectrum disorder and Rett syndrome compared to controls that implies that the microglia are activated. Additionally, an individual with Angelman syndrome and the sibling of an individual with autism have microglial densities similar to the individuals with autism and Rett syndrome. By contrast, an individual with Joubert syndrome, which is a developmental hypoplasia of the cerebellar vermis, had a normal density of microglia, indicating the specific pathology in the cerebellum does not necessarily result in increased microglial densities. We found a significant decrease in Purkinje cells specific to the cerebellar vermis in individuals with autism.

These findings indicate the importance for investigation of the Purkinje synapses in autism and that the relationship between the microglia and the synapses is of great utility in understanding the pathology in autism. Together, these data provide further evidence for the neuroinflammation hypothesis in autism and a basis for future investigation of neuroinflammation in autism. In particular, investigating the function of microglia in modifying synaptic connectivity in the cerebellum may provide key insights into developing therapeutics in autism spectrum disorder.

Firing Patterns of Cerebellar Purkinje Cells During Locomotion and Sleep

The cerebellum is a major supraspinal center involved in the coordination of movement. The principal neurons of the cerebellar cortex, Purkinje cells, receive excitatory synaptic input from two sources: the parallel and climbing fibers. These pathways have markedly different effects: the parallel fibers control the rate of simple sodium spikes, while the climbing fibers induce characteristic complex spike bursts, which are accompanied by dendritic calcium transients and play a key role in regulating synaptic plasticity. While many studies using a variety of species, behaviors, and cerebellar regions have documented modulation in Purkinje cell activity during movement, few have attempted to record from these neurons in unrestrained rodents. In this dissertation, we use chronic, multi-tetrode recording in freely-behaving rats to study simple and complex spike firing patterns during locomotion and sleep. Purkinje cells discharge rhythmically during stepping, but this activity is highly variable across steps. We show that behavioral variables systematically influence the step-locked firing rate in a step-phase-dependent way, revealing a functional clustering of Purkinje cells. Furthermore, we find a pronounced disassociation between patterns of variability driven by the parallel and climbing fibers, as well as functional differences between cerebellar lobules. These results suggest that Purkinje cell activity not only represents step phase within each cycle, but is also shaped by behavior across steps, facilitating control of movement under dynamic conditions. During sleep, we observe an attenuation of both simple and complex spiking, relative to awake behavior. Although firing rates during slow wave sleep (SWS) and rapid eye movement sleep (REM) are similar, simple spike activity is highly regular in SWS, while REM is characterized by phasic increases and pauses in simple spiking. This phasic activity in REM is associated with pontine waves, which propagate into the cerebellar cortex and modulate both simple and complex spiking. Such a temporal coincidence between parallel and climbing fiber activity is known to drive plasticity at parallel fiber synapses; consequently, pontocerebellar waves may provide a mechanism for tuning synaptic weights in the cerebellum during active sleep.

Morphological Alterations in Newly Born Dentate Gyrus Granule Cells That Emerge after Status Epilepticus Contribute to Make Them Less Excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.

Learning in the cerebellar nucleus is necessary for acquisition of Pavlovian eyelid conditioning

The cumulative work presented here supports the hypothesis that plasticity in the cerebellar cortex and cerebellar nuclei mediates a simple associative form of motor teaming-Pavlovian eyelid conditioning. It was previously demonstrated that focal ablative lesions of cerebellar anterior lobe or pharmacological block of the cerebellar cortex output disrupted the timing of the conditioned eyeblink response, unmasking a response with a relatively fixed and very short latency to onset. The results of this thesis demonstrate that the short-latency responses are due to associative learning. Unpaired training does not support the acquisition of short-latency responses while the rate of acquisition of short-latency responses during paired training is approximately the same as that of timed conditioned responses. The acquisition of short-latency responses is dependent on an intact cerebellar cortex. Both ablative lesions of the cerebellar cortex and inactivation of cerebellar cortex output with picrotoxin block the acquisition of short-latency responses. However, once the short-latency responses are acquired neither disconnection of cerebellar cortex nor inactivation of the cerebellar nucleus block reacquisition. The results are consistent with the proposal that plasticity in the cerebellar cortex is necessary for learning the timing of conditioned responses, plasticity in the interpositus nucleus mediates the short latency responses, and cerebellar cortical output and mossy fiber input are necessary for the acquisition of short latency responses. ^

Quantification of spread of cerebellar long-term depression with chemical two-photon uncaging of glutamate

Localized, chemical two-photon photolysis of caged glutamate was used to map the changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors caused by long-term synaptic depression (LTD) in cerebellar Purkinje cells. LTD produced by pairing parallel fiber activity with depolarization was accompanied by a decline in the response of Purkinje cells to uncaged glutamate that accounted for both the time course and magnitude of LTD. This depression of glutamate responses was observed not only at the site of parallel fiber stimulation but also at more distant sites. The amount of LTD decreased with distance and was half-maximal 50 μm away from the site of parallel fiber activity. Estimation of the number of parallel fibers active during LTD induction indicates that LTD modified glutamate receptors not only at active synapses but also at 600 times as many inactive synapses on a single Purkinje cell. Therefore, both active and inactive parallel fiber synapses can undergo changes at a postsynaptic locus as a result of associative pre- and postsynaptic activity.

Experimental diabetes in rats causes hippocampal dendritic and synaptic reorganization and increased glucocorticoid reactivity to stress

We report that 9 d of uncontrolled experimental diabetes induced by streptozotocin (STZ) in rats is an endogenous chronic stressor that produces retraction and simplification of apical dendrites of hippocampal CA3 pyramidal neurons, an effect also observed in nondiabetic rats after 21 d of repeated restraint stress or chronic corticosterone (Cort) treatment. Diabetes also induces morphological changes in the presynaptic mossy fiber terminals (MFT) that form excitatory synaptic contacts with the proximal CA3 apical dendrites. One effect, synaptic vesicle depletion, occurs in diabetes as well as after repeated stress and Cort treatment. However, diabetes produced other MFT structural changes that differ qualitatively and quantitatively from other treatments. Furthermore, whereas 7 d of repeated stress was insufficient to produce dendritic or synaptic remodeling in nondiabetic rats, it potentiated both dendritic atrophy and MFT synaptic vesicle depletion in STZ rats. These changes occurred in concert with adrenal hypertrophy and elevated basal Cort release as well as hypersensitivity and defective shutoff of Cort secretion after stress. Thus, as an endogenous stressor, STZ diabetes not only accelerates the effects of exogenous stress to alter hippocampal morphology; it also produces structural changes that overlap only partially with those produced by stress and Cort in the nondiabetic state.

Steel mutant mice are deficient in hippocampal learning but not long-term potentiation.

Mice carrying mutations in either the dominant white-spotting (W) or Steel (Sl) loci exhibit deficits in melanogenesis, gametogenesis, and hematopoiesis. W encodes the Kit receptor tyrosine kinase, while Sl encodes the Kit ligand, Steel factor, and the receptor-ligand pair are contiguously expressed at anatomical sites expected from the phenotypes of W and Sl mice. The c-kit and Steel genes are also both highly expressed in the adult murine hippocampus: Steel is expressed in dentate gyrus neurons whose mossy fiber axons synapse with the c-kit expressing CA3 pyramidal neurons. We report here that Sl/Sld mutant mice have a specific deficit in spatial learning. These mutant mice are also deficient in baseline synaptic transmission between the dentate gyrus and CA3 but show normal long-term potentiation in this pathway. These observations demonstrate a role for Steel factor/Kit signaling in the adult nervous system and suggest that a severe deficit in hippocampal-dependent learning need not be associated with reduced hippocampal long-term potentiation.

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.