Raymund Y. K. Pun

Pilocarpine-Induced Seizures Cause Selective Time-Dependent Changes to Adult-Generated Hippocampal Dentate Granule Cells

Aberrantly interconnected granule cells are characteristic of temporal lobe epilepsy. By reducing network stability, these abnormal neurons may contribute directly to disease development. Only subsets of granule cells, however, exhibit abnormalities. Why this is the case is not known. Ongoing neurogenesis in the adult hippocampus may provide an explanation. Newly generated granule cells may be uniquely vulnerable to environmental disruptions relative to their mature neighbors. Here, we determine whether there is a critical period after neuronal birth during which neuronal integration can be disrupted by an epileptogenic insult. By bromodeoxyuridine birthdating cells in green fluorescent protein-expressing transgenic mice, we were able to noninvasively label granule cells born 8 weeks before (mature), 1 week before (immature), or 3 weeks after (newborn) pilocarpine-epileptogenesis. Neuronal morphology was examined 4 and 8 weeks after pilocarpine treatment. Strikingly, almost 50% of immature granule cells exposed to pilocarpine-epileptogenesis exhibited aberrant hilar basal dendrites. In contrast, only 9% of mature granule cells exposed to the identical insult possessed basal dendrites. Moreover, newborn cells were even more severely impacted than immature cells, with 40% exhibiting basal dendrites and an additional 20% exhibiting migration defects. In comparison, <5% of neurons from normal animals exhibited either abnormality, regardless of age. Together, these data demonstrate the existence of a critical period after the birth of adult-generated neurons during which they are vulnerable to being recruited into epileptogenic neuronal circuits. Pathological brain states therefore may pose a significant hurdle for the appropriate integration of newly born endogenous, and exogenous, neurons.

Deterministic center of pressure patterns characterize postural instability in Parkinson's disease

Static posturographic recordings were obtained from six Parkinson’s patients and six age-matched, healthy control participants. The availability of vision and visuo-spatial cognitive load were manipulated. Postural sway patterns were analyzed using recurrence quantification analysis (RQA), which revealed differences in center of pressure (COP) dynamics between Parkinson’s and control participants. AP COP trajectories for the Parkinson’s group were not only significantly more variable than for the control group, but also exhibited distinct patterns of temporal dynamics. The visual manipulation did not differentially affect the two groups. No cognitive load effects were found. The results are generally consistent with the hypothesis that pathological physiological systems exhibit a tendency for less flexible, more deterministic dynamic patterns.

Synaptic excitation in cultures of mouse spinal cord neurones: receptor pharmacology and behaviour of synaptic currents.

Fast monosynaptic excitatory post‐synaptic potentials between spinal cord neurones in cell culture (s.c.‐s.c. e.p.s.p.s) were studied with current‐clamp and two‐electrode voltage‐clamp methods. The reversal potential, response to acidic amino acid antagonists, and behaviour of the synaptic current were examined. The amplitude of the e.p.s.p. increased with membrane potential hyperpolarization and decreased with depolarization. The reversal potential of the e.p.s.p. was +3.8 +/‐ 2.5 mV (mean +/‐ S.E. of mean). The reversal potential for responses to ionophoretically applied L‐glutamate and L‐aspartate was also near 0 mV. The acidic amino acid antagonist, cis‐2,3‐piperidine dicarboxylic acid (PDA, 0.25‐1.0 mM) reversibly antagonized the monosynaptic e.p.s.p.s as well as responses to kainate (KA) or quisqualate (QA). The selective N‐methyl‐D‐aspartate antagonist, (+/‐) 2‐amino‐5‐phosphonovaleric acid (APV), had little effect on either the monosynaptic e.p.s.p.s or responses to QA or KA at concentrations that abolished responses to L‐aspartate. Under voltage clamp, the peak synaptic current (e.p.s.c.) was linearly related to the membrane potential, increasing in amplitude with hyperpolarization and decreasing with depolarization from the resting potential. The decay of a somatic e.p.s.c. was well fitted by a single exponential function with a time constant of 0.6 ms at 25 degrees C. E.p.s.c.s which had proximal dendritic locations had decay time constants of 1‐2 ms. The decay time constant was voltage‐insensitive between ‐80 and +10 mV. We suggest that an acidic amino acid receptor other than that for NMDA mediates excitatory transmission at the s.c.‐s.c. synapse; and that the underlying conductance mechanism is voltage insensitive with an estimated mean channel lifetime of less than 1 ms.

Heterogeneous integration of adult-generated granule cells into the epileptic brain

The functional impact of adult-generated granule cells in the epileptic brain is unclear, with data supporting both protective and maladaptive roles. These conflicting findings could be explained if new granule cells integrate heterogeneously, with some cells taking neutral or adaptive roles and others contributing to recurrent circuitry supporting seizures. Here, we tested this hypothesis by completing detailed morphological characterizations of age- and experience-defined cohorts of adult-generated granule cells from transgenic mice. The majority of newborn cells exposed to an epileptogenic insult exhibited reductions in dendritic spine number, suggesting reduced excitatory input to these cells. A significant subset, however, exhibited higher spine numbers. These latter cells tended to have enlarged cell bodies, long basal dendrites, or both. Moreover, cells with basal dendrites received significantly more recurrent mossy fiber input through their apical dendrites, indicating that these cells are robustly integrated into the pathological circuitry of the epileptic brain. These data imply that newborn cells play complex—and potentially conflicting—roles in epilepsy.

Adenosine modulates hypoxia‐induced responses in rat PC12 cells via the A2A receptor

1 The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen‐sensitive clonal cell line. 2 Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3 Whole‐cell current‐ and voltage‐clamp experiments showed that adenosine attenuated the hypoxia‐induced membrane depolarization. The hypoxia‐induced suppression of the voltage‐sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration‐dependent manner. This increase was blocked by pretreatment not only with a non‐specific adenosine receptor antagonist, 8‐phenyltheophylline (8‐PT), but also with a selective A2A receptor antagonist, ZM241385. 4 Ca2+ imaging studies using fura‐2 acetoxymethyl ester (fura‐2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage‐clamp studies showed that adenosine inhibited the voltage‐dependent Ca2+ currents (ICa) in a concentration‐dependent fashion. This inhibition was also abolished by both 8‐PT and ZM241385. 5 The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6‐22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA‐deficient PC12 cells. 6 These results indicate that the modulatory effects of adenosine on the hypoxia‐induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and possibly other oxygen‐sensitive cells during hypoxia.

G-protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells.

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca2+ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 μM GTP-γS did not significantly affect prepulse facilitation or whole-cell Ba2+ current (IBa) density. In contrast, inactivation of G proteins by intracellular GDP-βS or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of IBa, respectively. GDP-βS dialysis resulted in nearly a threefold increase in peak IBa density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 μm intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca2+ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (∼50%) in IBa amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca2+ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.

Activation of serotonin1A receptors inhibits midbrain periaqueductal gray neurons of the rat.

The midbrain periaqueductal gray (PAG) is involved in a variety of functions including pain modulation, vocalization, autonomic control, fear and anxiety. This area contains serotonin receptors, particularly 5-HT1A that are known to play a role in the above functions. The goals of this study were to characterize the effects of 8-OH-DPAT, a selective 5-HT1A agonist, on the firing characteristics and membrane properties of PAG neurons. Both in vivo and in vitro preparations were used. The effects of 8-OH-DPAT on baseline activity of 91 neurons were tested in the in vivo preparation. In 50/91 cells, 8-OH-DPAT produced a decrease in the firing rate that ranged between 21 and 98% (mean +/- S.E.M. decrease of 49 +/- 1.9%). This inhibitory effect was dose dependent and could be blocked by spiperone. In 10/91 cells, 8-OH-DPAT produced an increase in the firing rate that ranged between 13 and 290%, with mean increase of 83 +/- 7.4%. The baseline firing rate of the remaining 31 cells was not affected by 8-OH-DPAT. In the PAG slice preparation, the effects of 8-OH-DPAT on synaptic and membrane properties of 17 PAG neurons were tested using whole-cell voltage clamp-recording procedures. In 14 cells, application of 8-OH-DPAT produced hyperpolarization that ranged between 6 and 21 mV, with mean of 8.4 +/- 2.0 mV. This hyperpolarization was associated with a decrease in membrane impedance that ranged between 8 and 45%, with mean decrease of 21.6 +/- 4.5%. The remaining three neurons did not respond to 8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured rat olfactory neurons are excitable and respond to odors.

Newborn rat nasal tissues containing olfactory epithelium were dissociated and maintained in a monolayer cell culture. Neurons were present, as determined by immunostaining with antibodies to 4 neuron-specific proteins: neuron-specific enolase, microtubule-associated protein 2, tau protein and synaptophysin. Immunostained neurons had a distinctive morphology resembling olfactory neurons. By patch-clamp analysis, these cells were electrically active. Responses of some neurons to physiological concentrations of an odorant mixture identified them as olfactory receptor cells.

Impact of corticosterone treatment on spontaneous seizure frequency and epileptiform activity in mice with chronic epilepsy.

Stress is the most commonly reported precipitating factor for seizures in patients with epilepsy. Despite compelling anecdotal evidence for stress-induced seizures, animal models of the phenomena are sparse and possible mechanisms are unclear. Here, we tested the hypothesis that increased levels of the stress-associated hormone corticosterone (CORT) would increase epileptiform activity and spontaneous seizure frequency in mice rendered epileptic following pilocarpine-induced status epilepticus. We monitored video-EEG activity in pilocarpine-treated mice 24/7 for a period of four or more weeks, during which animals were serially treated with CORT or vehicle. CORT increased the frequency and duration of epileptiform events within the first 24 hours of treatment, and this effect persisted for up to two weeks following termination of CORT injections. Interestingly, vehicle injection produced a transient spike in CORT levels – presumably due to the stress of injection – and a modest but significant increase in epileptiform activity. Neither CORT nor vehicle treatment significantly altered seizure frequency; although a small subset of animals did appear responsive. Taken together, our findings indicate that treatment of epileptic animals with exogenous CORT designed to mimic chronic stress can induce a persistent increase in interictal epileptiform activity.

Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca2+ current (ICa) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p≤0.05). Analysis of voltage-dependent steady-state activation (m∞) curves revealed a 0.70±0.27 charge increase in the activation-gate valency (zm) following 8-Br-cAMP perfusion. Similar responses were observed when Ba2+ was the charge carrier, where zm was increased by 1.33±0.34 charges (n=8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for IBa (mean, n=8). The time course for IBa (and ICa) activation was well described by second-order m2 kinetics. The derived time constant for activation (τm) was voltage-dependent, and the τm/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the (τm) and m∞2values according to a Hodgkin-Huxley type m2 activation process. The forward rate (αm) for channel activation was increased by 8-Br-cAMP at membrane potentials ≥0 mV, and the backward rate (βm) decreased at potentials ≤ +10 mV. Time-dependent inactivation of ICa consisted of a slowly decaying component (τh ≈ 300 ms) and a “non-inactivating” steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.