Jean C. Strahlendorf

Facilitation of the lumbar monosynaptic reflexes by locus coeruleus stimulation

Abstract A conditioning train of stimuli delivered in the vicinity of the locus coeruleus elicited a marked (average 104%) facilitation of the lumbar monosynaptic reflex in decerebrated cats. Placement of the electrode in periaqueductal gray resulted in a biphasic facilitatory action in the MSR. Phenoxybenzamine (2 mg/kg) and chlorpromazine (1–2 mg/kg) caused marked reductions in LC-induced augmentations. Haloperidol appeared to potentiate LC-elicited facilitation. These data further verify the noradrenergic nature of fibers originating from the locus coeruleus and assign a physiological role to coeruleo-spinal projection in the regulation of lumbar MSR.

Modulatory Action of serotonin on glutamate-induced excitation of cerebellar purkinje cells

The effects of serotonin (5-hydroxytryptamine, 5-HT) on glutamate-induced excitation of Purkinje cells were examined. Pulsatile iontophoretic applications of glutamate (1-25 nA) induced consistent increases in the spontaneous activity of Purkinje cells. When serotonin was applied continuously with currents that elicited minimum changes in the spontaneous rate, it inhibited or blocked glutamate-induced excitations significantly in most Purkinje cells. We also examined the effects of high currents of serotonin on spontaneous activity of Purkinje cells. High currents of serotonin induced 3 different effects: inhibitions, biphasic effects comprising transient inhibition followed by excitation, and excitations. Nonetheless, whether it inhibited or excited the activity of Purkinje cells, serotonin inhibited glutamate-induced excitations consistently. The effect of a putative 5-HT antagonist methysergide (UML) was also examined. Methysergide consistently attenuated or antagonized the inhibitory effects of serotonin on glutamate-induced excitations. This finding suggests strongly that inhibitory effects of 5-HT on glutamate excitations observed in the present study is the specific action of serotonin.

Effects of serotonin on cerebellar Purkinje cells are dependent on the baseline firing rate

SummarySerotonin applied iontophoretically to cerebellar Purkinje cells elicited one of three effects: inhibition (62% of the cells), biphasic response (27%), and excitation (11%). This study describes a correlation between the spontaneous discharge rate of Purkinje cells and the action of serotonin. Purkinje neurons that responded to serotonin with an increase in firing rate had a significantly slower predrug firing frequency (40 Hz) than those cells that were suppressed by serotonin (51 Hz). Furthermore, it was shown that with increasing firing rates the proportion of excitations decreased, and the proportion of depressions increased. A quantitative comparison revealed a statistically significant correlation between the spontaneous discharge rate of cells displaying excitation and the magnitude of the excitatory response. On several occasions, the direction of the Purkinje cell response to serotonin reversed following a decrease or increase in the baseline spontaneous rate. Glutamate- or asparate-induced excitations elicited an augmentation of serotonin-mediated inhibition and in some cases a reversal of excitation to inhibition. Likewise, the lowering of neuronal activity by the continuous application of glycine augmented excitation and reduced and reversed serotonin inhibitions. Preliminary results from experiments in which various receptor antagonists were tested against serotonin actions suggest that the effects of serotonin occur, at least in part, at postsynaptic sites on Purkinje cells. These results strongly suggest that the overall qualitative effects of serotonin is to set Purkinje cells at a preferred firing rate. In this sense, the term biaser or modulator may best describe the role of serotonin in the cerebellum.

Active caspase-3 expression during postnatal development of rat cerebellum is not systematically or consistently associated with apoptosis

Development is a dynamic process that includes an intricate balance between an increase in cell mass and an elimination of excess or defective cells. Although caspases have been intimately linked to apoptotic events, there are a few reports suggesting that these cysteine proteases can influence the differentiation and proliferation of cells. Specifically, the active form of caspase‐3, which has been classified as an executor of apoptosis, recently has been implicated in a nonapoptotic role in the regulation of the cell cycle, cell proliferation, and cell differentiation. This study investigated the nonapoptotic function and phenotypic expression of active caspase‐3‐positive cells in the external granule cell layer (EGL) of the postnatal rat cerebellum by using biochemical and immunohistochemical analyses, respectively. Evidence that negates an apoptotic function for the caspase‐3‐positive EGL cells includes a failure to exhibit chromatin condensation (assessed with TOPRO), phosphatidyl serine externalization (Annexin V labeling), or DNA fragmentation (TUNEL labeling). Proliferative (Ki67‐positive) and differentiated (TUJ1‐positive) cells within the EGL exhibited a cytosolic expression of caspase‐3, whereas terminally differentiated granule cells (NeuN‐positive) in the internal granular layer and the migrating granule cells did not express active caspase‐3. Thus, this study supports a nonapoptotic role for active caspase‐3 in cells residing in the EGL and suggests a possible involvement in EGL proliferation and differentiation. J. Comp. Neurol. 476:154–173, 2004.

Response of cerebellar Purkinje cells to serotonin and the 5-HT1A agonists 8-OH-DPAT and ipsapirone in vitro

These studies were undertaken in an attempt to classify the receptor subtypes mediating the inhibitory effects of serotonin (5-HT) on cerebellar Purkinje cells in the in vitro slice preparation. 5-HT and the 5-HT1A specific agonists 8-hydroxy-2(di-n-propyl-amino)tetralin (8-OH-DPAT) and ipsapirone were iontophoretically applied to Purkinje cells during control periods and periods of concurrent application of the 5-HT1A/5-HT2 antagonist spiperone. 5-HT was found to produce three distinct effects on Purkinje cell spontaneous discharge: inhibition, excitation and a biphasic effect. Iontophoretically applied 8-OH-DPAT and ipsapirone elicited only inhibition of Purkinje cell firing in all cells tested. Purkinje cell inhibitions elicited by 5-HT, 8-OH-DPAT and ipsapirone were all found to be significantly dose-dependent. However, while dose-response curves for 8-OH-DPAT and ipsapirone were found to be identical, they both differed significantly from the 5-HT curve. Spiperone was shown to significantly attenuate Purkinje cell inhibition induced by 5-HT, 8-OH-DPAT and ipsapirone. In several cells 5-HT-induced inhibition of spontaneous discharge was reversed to excitation in the presence of spiperone. This was never observed with either 8-OH-DPAT or ipsapirone. Thus, our results suggest that 5-HT-induced Purkinje cell inhibitions are at least partially mediated by the 5-HT1A receptor subtype, and there also may be additional 5-HT receptor subtypes present mediating other responses. Ultimate Purkinje cell responses to 5-HT may be due to summation of responses induced by activation of several 5-HT receptor subtypes.

Bergmann glia utilize active caspase-3 for differentiation

Recently, functions associated with caspase have been modified from their well-established role in apoptosis. Although caspases are still regarded as mediators of apoptosis, some of the pro-apoptotic caspases, namely caspase-8, -14 and -3 also regulate differentiation in certain cell types, namely myelomonocytic cells, osteoblasts, skeletal muscle cells, keratinocytes, and T lymphocytes. In the central nervous system, non-apoptotic active caspase-3 expression has been located in proliferating and differentiating neuronal cells of the ventricular zone and external granular layer of the developing cerebellar cortex. We previously demonstrated that active caspase-3 expression was not limited to neuronal cells but also was located in the Bergmann glia of the postnatal cerebellum. In that study, active caspase-3 immunolabeling did not markedly colocalize with Ki67, a proliferation marker, but was present in differentiating Bergmann glia that expressed brain lipid binding protein (BLBP) and thus, by its localization, suggested a role in the differentiation of Bergmann glia. The current study addresses the function of caspase-3 in Bergmann glia development by utilizing a Bergmann glial culture preparation. Inhibition of caspase-3 activity by the peptide inhibitor, DMQD-FMK, increased the number of proliferating precursor glial cells and decreased the number of differentiating Bergmann glia, without significantly altering the non-glial active caspase-3 negative population. The transformation in the developmental state of Bergmann glia occurring after suppression of caspase-3 activity strongly suggests an involvement of this enzyme in promoting differentiation of Bergmann glia.

Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons.

In the rat cerebellar slice preparation, exposure to hypoxia elicited by a 30 min exposure to artificial cerebrospinal fluid continuously gassed with 95% N(2): 5% CO(2) induced a characteristic type of toxicity of Purkinje cells (PCs) resembling excitotoxic-mediated dark cell degeneration (DCD). Morphologically, PCs exhibited marked rounded appearance with cytoplasmic darkening, nuclear condensation and cytoplasmic vacuoles. Using gel electrophoresis, genomic DNA obtained from the cerebellar slice exhibited fragmentation. However, PCs failed to exhibit apoptotic bodies or evidence of phagocytosis, spherical- or crescent-shaped chromatin aggregations or TUNEL-positive staining. Ultrastructural analyses of granule cells revealed the presence of apoptotic bodies and discrete spherical collection of chromatin clumping as well as phagocytosis suggesting that the oligonucleosomal-sized DNA fragments primarily were derived from granule cells. PC-elicited toxicity was attenuated significantly in the presence of the competitive AMPA and NMDA antagonists CNQX and APV, respectively. The present study extends the involvement of excitotoxic processes in mediating hypoxic-induced toxicity of PCs in postnatal rats and suggests, in contrast to DCD elicited by direct application of excitotoxic agents, that DCD associated with acute hypoxic insults in PCs does not resemble classical apoptosis.

Serotonin alters an inwardly rectifying current (Ih) in rat cerebellar Purkinje cells under voltage clamp

The effects of serotonin (5-HT) on the hyperpolarization-activated sodium-potassium inward current (H-current: Ih) were examined in cerebellar Purkinje cells (PCs) under current- and voltage-clamp conditions. Quasi-steady state current versus voltage relationships under voltage clamp conditions showed that 5-HT decreased the inward rectification at potentials negative to -70 mV with a corresponding decrease in the slope conductance. In 70% of PCs, 5-HT produced dose-related attenuations of the Ih current with corresponding decreases in slope conductances across a range of hyperpolarized potentials. A negative shift in the Ih activation curve was elicited by 5-HT so that the amplitude of Ih current active near rest and at more hyperpolarized ranges was decreased. Serotonin-induced inhibition of Ih shows some receptor subtype selectivity in that DOI, the 5-HT2/IC receptor agonist, most closely mimicked the actions of 5-HT. This study reveals a novel inhibitory action of 5-HT on Ih in cerebellar Purkinje cells which may contribute to direct inhibitory effects of 5-HT on spontaneous firing and modulatory actions on gamma-aminobutyric acid (GABA)-mediated responses to PCs.

A transient voltage-dependent outward potassium current in mammalian cerebellar Purkinje cells

Utilizing the single electrode voltage clamp technique applied to rat Purkinje cells (PCs), we have recorded a transient outward voltage-dependent potassium current, Ito. Half maximal values for inactivation and activation were -65 mV and -39 mV, respectively. Ito decays as a single exponential with a time constant of 95 ms and is reduced by 4-aminopyridine. Based on criteria of voltage dependency of activation and inactivation, kinetics of inactivation, ionic selectivity and pharmacologic sensitivity, we have verified a strong resemblance between the typical A current in other neurons, and Ito in PCs. As in other cells, Ito may be important in shaping PC output by modulating intrinsic and extrinsic factors that govern PC firing.

Modulatory role of serotonin on GABA-elicited inhibition of cerebellar Purkinje cells

The present study was designed to directly examine the postsynaptic actions of serotonin on GABA-mediated inhibition of cerebellar Purkinje cells. The findings indicate that serotonin at currents that produced minimal effects on the spontaneous firing rates of Purkinje cells modified GABA effects in a biphasic manner. Serotonin initially decreased GABA-mediated inhibitions followed secondarily by either continued inhibition or, in the majority of cases, augmentation of GABA responses. When a comparison was made of the secondary effects of serotonin on GABA-mediated inhibition with the initial spontaneous firing rates of the Purkinje cells, the group in which serotonin augmented GABA actions had a significantly higher initial firing frequency than the group in which serotonin attenuated GABA-mediated inhibition. Furthermore, with increasing firing rates, the proportion of cells showing augmentation of GABA inhibition increased, and the proportion of cells displaying attenuation of GABA effects decreased. Serotonin affected beta-alanine-mediated inhibitions in a manner similar to that seen with GABA, whereas glycine was differentially altered. This study identifies another neuromodulatory role of serotonin on Purkinje cells in the cerebellum. Furthermore, the effects of serotonin on GABA inhibition seem to be governed by some intrinsic property of the Purkinje cell, which is apparently related to the firing rate of the cell.