Surviving mossy cells enlarge and receive more excitatory synaptic input in a mouse model of temporal lobe epilepsy

Numerous hypotheses of temporal lobe epileptogenesis have been proposed, and several involve hippocampal mossy cells. Building on previous hypotheses we sought to test the possibility that after epileptogenic injuries surviving mossy cells develop into super‐connected seizure‐generating hub cells. If so, they might require more cellular machinery and consequently have larger somata, elongate their dendrites to receive more synaptic input, and display higher frequencies of miniature excitatory synaptic currents (mEPSCs). To test these possibilities pilocarpine‐treated mice were evaluated using GluR2‐immunocytochemistry, whole‐cell recording, and biocytin‐labeling. Epileptic pilocarpine‐treated mice displayed substantial loss of GluR2‐positive hilar neurons. Somata of surviving neurons were 1.4‐times larger than in controls. Biocytin‐labeled mossy cells also were larger in epileptic mice, but dendritic length per cell was not significantly different. The average frequency of mEPSCs of mossy cells recorded in the presence of tetrodotoxin and bicuculline was 3.2‐times higher in epileptic pilocarpine‐treated mice as compared to controls. Other parameters of mEPSCs were similar in both groups. Average input resistance of mossy cells in epileptic mice was reduced to 63% of controls, which is consistent with larger somata and would tend to make surviving mossy cells less excitable. Other intrinsic physiological characteristics examined were similar in both groups. Increased excitatory synaptic input is consistent with the hypothesis that surviving mossy cells develop into aberrantly super‐connected seizure‐generating hub cells, and soma hypertrophy is indirectly consistent with the possibility of axon sprouting. However, no obvious evidence of hyperexcitable intrinsic physiology was found. Furthermore, similar hypertrophy and hyper‐connectivity has been reported for other neuron types in the dentate gyrus, suggesting mossy cells are not unique in this regard. Thus, findings of the present study reveal epilepsy‐related changes in mossy cell anatomy and synaptic input but do not strongly support the hypothesis that mossy cells develop into seizure‐generating hub cells. © 2014 Wiley Periodicals, Inc.     

In vivo two‐photon imaging of structural dynamics in the spinal dorsal horn in an inflammatory pain model

Two‐photon microscopy imaging has recently been applied to the brain to clarify functional and structural synaptic plasticity in adult neural circuits. Whereas the pain system in the spinal cord is phylogenetically primitive and easily exhibits behavioral changes such as hyperalgesia in response to inflammation, the structural dynamics of dendrites has not been analysed in the spinal cord mainly due to tissue movements associated with breathing and heart beats. Here we present experimental procedures to prepare the spinal cord sufficiently to follow morphological changes of neuronal processes in vivo by using two‐photon microscopy and transgenic mice expressing fluorescent protein specific to the nervous system. Structural changes such as the formation of spine‐like structures and swelling of dendrites were observed in the spinal dorsal horn within 30 min after the multiple‐site injections of complete Freund's adjuvant (a chemical irritant) to a leg, and these changes continued for 5 h. Both AMPA and N‐methyl‐D‐aspartate receptor antagonists, and gabapentin, a presynaptic Ca²⁺ channel blocker, completely suppressed the inflammation‐induced structural changes in the dendrites in the spinal dorsal horn. The present study first demonstrated by in vivo two‐photon microscopy imaging that structural synaptic plasticity occurred in the spinal dorsal horn immediately after the injection of complete Freund's adjuvant and may be involved in inflammatory pain. Furthermore, acute inflammation‐associated structural changes in the spinal dorsal horn were shown to be mediated by glutamate receptor activation.     

Autoantibody against dendrite in Plasmodium falciparum infection: a singular auto-immune phenomenon preferentially in cerebral malaria

To investigate auto-reactive antibodies against dendrites of neurons (AAD) previously reported in cerebral malaria (CM) for their functional biological activity, a serological study was conducted in a larger cohort of patients with CM and uncomplicated falciparum malaria (UM). Sera from Thai adults with CM (n = 22) and UM (n = 21) were tested to determine the titers of AAD by indirect fluorescent antibody test and specific antibody responses to Plasmodium falciparum antigens by ELISA. Immunoreactivity against the dendrites of neurons was observed in 100% of sera from the cerebral malaria group as compared to 71% from the non-cerebral malaria group, and the median titer of AAD was higher in CM versus UM, though the difference did not reach significance. In contrast an opposite pattern was seen for anti-P. falciparum antibody titers, which were significantly lower among CM than among UM patients, both for IgG and IgM (p = 0.024 and p = 0.0033, respectively). Our results indicate that this auto-immune phenomenon induced by P. falciparum infection occurs preferentially in cerebral malaria despite lower responses in parasite-specific antibody responses.     

Activity-dependent dendritic spine neck changes are correlated with synaptic strength

Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d’etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons.Dendritic spines are found in neurons throughout the central nervous system (1), and in pyramidal neurons receive the majority of excitatory inputs, whereas dendritic shafts are normally devoid of glutamatergic synapses (2–7). These facts suggest that spines are likely to play an essential role in neural circuits (1), although it is still unclear exactly what this role is (8, 9). Because of their peculiar morphology, hypotheses regarding the specific function of spines have focused on their role in biochemical compartmentalization, whereby a small spine head, where the excitatory synapse is located, is separated from the parent dendrite by a thin neck, isolating the spine cytoplasm from the dendrite (10). Indeed, spines are diffusionally restricted from dendrites (11–13) and compartmentalize calcium after synaptic stimulation (14–16). This local biochemistry and the high calcium accumulations observed following temporal pairing of neuronal input and output (14, 17, 18) are thought to be responsible for input-specific synaptic plasticity (19–21). However, besides this biochemical role, spines have also been hypothesized to play an electrical role, altering excitatory postsynaptic potentials (EPSPs) (22–30). Consistent with this idea, activating spines with two-photon uncaging of glutamate generates potentials whose amplitudes are inversely proportional to the length of the spine neck (31), and these responses are much larger in spines than in adjacent dendritic shafts (32). Also, spine conductances can be activated independently of dendritic ones (33–36). These data suggest that spines could serve as electrical compartments but, at the same time, raise the issue of the functional significance of the thousands of long-necked spines that cover the dendrites of pyramidal neurons, which would therefore have negligible somatic voltage contributions.In this study we first undertook a series of experiments to discern the potential effect that the spine neck length has on the synaptic potentials generated by minimal synaptic stimulation at identified spines. We find that EPSP amplitudes are inversely correlated with spine neck lengths and that, as also seen in glutamate uncaging experiments (31), long-necked spines do not appear to generate any significant somatic depolarizations. In a separate set of experiments, we used a spike timing-dependent long-term potentiation (STD-LTP) induction protocol to trigger rapid shortening of the stimulated spine neck, which was accompanied by increases in the amplitude of the evoked potentials. In essence, we thus found a way to rapidly increase the voltage contribution of long-necked spines. To dissect the plausible mechanisms of the effect, we conducted biophysical simulations in the software NEURON. Our models show that the observed phenomenon could be accounted for by rapid regulation of synaptic conductance or, alternatively, stem from electrical attenuation effects due to the changes in spine neck resistance associated with changes in neck length. The spine neck resistance values necessary to entirely account for such attenuation are at odds with reported estimates (13, 32), so one would be inclined to assume that a rapid increase in synaptic conductance leads to the observed changes in somatic EPSP size. However, because spine neck resistance values have so far been inferred only indirectly, one cannot rule out the possibility that a combination of (synaptic) conductance and (neck) resistance changes could contribute to the observed activity-dependent changes in somatic EPSP size.     

Corticosterone reduces dendritic complexity in developing hippocampal CA1 neurons

Although prolonged stress and corticosteroid exposure induce morphological changes in the hippocampal CA3 area, the adult CA1 area is quite resistant to such changes. Here we addressed the question whether elevated corticosteroid hormone levels change dendritic complexity in young, developing CA1 cells. In organotypic cultures (prepared from P5 rats) that were 14–21 days cultured in vitro, two doses of corticosterone (30 and 100 nM) were tested. Dendritic morphology of CA1 neurons was established by imaging neurons filled with the fluorescent dye Alexa. Application of 100 nM corticosterone for 20 minutes induced atrophy of the apical dendritic tree 1–4 hours later. Fractal analysis showed that total neuronal complexity was reduced twofold when compared with vehicle‐treated neurons. Exposing organotypic slices to 30 nM corticosterone reduced apical length in a more delayed manner: only neurons examined more than 2 hours after exposure to corticosterone showed atrophy of the apical dendritic tree. Neither dose of corticosterone affected the length of basal dendrites or spine density. Corticosterone was ineffective in changing morphology of the apical dendrites when tested in the presence of the glucocorticoid receptor antagonist RU38486. These results suggest that high physiological levels of corticosterone, via activation of the glucocorticoid receptor, can, during the course of only a few hours, reduce the dendritic complexity of CA1 pyramidal neurons in young, developing hippocampal tissue. These findings suggest that it is relevant to maintain plasma corticosterone levels low during hippocampal development. © 2009 Wiley‐Liss, Inc.     

Distinct nonuniform cable properties optimize rapid and efficient activation of fast-spiking GABAergic interneurons

Fast-spiking, parvalbumin-expressing basket cells (BCs) play a key role in feedforward and feedback inhibition in the hippocampus. However, the dendritic mechanisms underlying rapid interneuron recruitment have remained unclear. To quantitatively address this question, we developed detailed passive cable models of BCs in the dentate gyrus based on dual somatic or somatodendritic recordings and complete morphologic reconstructions. Both specific membrane capacitance and axial resistivity were comparable to those of pyramidal neurons, but the average somatodendritic specific membrane resistance (R_m) was substantially lower in BCs. Furthermore, R_m was markedly nonuniform, being lowest in soma and proximal dendrites, intermediate in distal dendrites, and highest in the axon. Thus, the somatodendritic gradient of R_m was the reverse of that in pyramidal neurons. Further computational analysis revealed that these unique cable properties accelerate the time course of synaptic potentials at the soma in response to fast inputs, while boosting the efficacy of slow distal inputs. These properties will facilitate both rapid phasic and efficient tonic activation of BCs in hippocampal microcircuits.     

UVB照射对PIG1细胞增殖活性的影响

目的：探讨UVB照射对正常人黑素细胞PIG1细胞形态及增殖活性的影响。方法：取对数生长期的PIG1细胞,分别以50、100、200、300、400、500、600和700mJ/cm2剂量的UVB照射后继续培养0h、24h和48h,在倒置显微镜下观察其形态学变化,用MTT法检测UVB照射对细胞增殖活性的影响。结果：在50～200 mJ/cm2的UVB辐射下,随辐射剂量的增大,PIG1细胞的增殖活性逐渐增加,倒置相差显微镜从形态学上证实了一定剂量的UVB照射可以促进细胞增殖。结论：一定剂量的UVB照射有刺激PIG1细胞增殖及树突生长的作用。     

Altered morphology of motor cortex neurons in the VPA rat model of autism

Autism occurs in 1 in 1,000 children and incidence may be increasing. Investigating brain development and developmental injury in humans is difficult. As such, many studies rely on animal models of disorders. We chose to investigate the valproic acid-exposed rat, as this model shares many similarities with autism. Pregnant Long-Evans rats were administered either valproic acid (VPA) or saline during fetal neural tube development. Morphological analyses of cells in layer II of the golgi impregnated motor cortex were done to determine dendritic length, volume, and complexity in both groups. No differences were found in length or volume of cortical dendrites, but dendritic arborization was more complex in apical dendrites of pyramidal cells in VPA-exposed animals than controls. The implication of this finding is that pruning in the VPA-exposed rat is not occurring, which is consistent with theories related to abnormal human brain development in autism.     

Multiple actions of iontophoretically applied serotonin on motorneurones in the turtle spinal cord in vitro

The effects of focal activation of serotonergic receptors in motorneurones were investigated in a slice preparation of the turtle spinal cord. The test response to glutamate evoked from a dendrite by iontophoresis was attenuated by serotonin or 8-hydroxy-dipropyl-aminotetralin (8-OH-DPAT) applied from an independent pipette within 100 μm of the glutamate pipette. This effect was not associated with a conductance change and did not affect glutamate responses evoked more than 100 μm from the serotonin pipette. The effect of serotonin was not reproduced by H⁺ ions. Plateau potentials were rarely facilitated by serotonin when applied in the dendritic field. Plateau potentials were readily facilitated by serotonin applied near the soma. This effect was preceded by attenuation of the slow after-hyperpolarization following an action potential. Applied near the soma, serotonin inhibited the response to a depolarizing current pulse of moderate strength and enhanced the response to a current pulse of high amplitude. These effects were associated with a conductance increase. We conclude that serotonin has spatially diverse effects on motorneurones.     

Ultrastructural observations on beaded α-motoneuron dendrites

Beaded dendrites of 1α-motoneurons intracellularly labelled with horseradish peroxidase (HRP) were studied ultrastructurally in eight adult cats. For comparison, adjacent unlabelled beaded dendrites of unknown origin were also included in the study. Electron microscopy revealed no signs of degeneration or poor fixation according to common criteria. With the exception of the HRP-reaction product no difference in structure was observed between labelled and unlabelled beaded dendrites. Both the beads and their interconnecting segments were postsynaptic to boutons of normal appearance containing spherical (S-type boutons) or flattened vesicles (F-type boutons). The values for synaptic covering and synaptic packing density of the beaded dendritic regions, which usually were located in the periphery of the dendritic trees, were clearly lower than values obtained previously for cell bodies and proximal dendrites of a-motoneurons.