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Congenitally anophthalmic mice (ZRDCT-AN) have circadian rhythms which 'free-run' and are not light modulated. Their rhythms differ from those of controls in: duration of circadian period, length of active phase, and pattern/intensity of activity. Three different populations have been described based upon wheel-running: rhythmic with stable period, rhythmic with unstable period and arrhythmic. Circadian rhythms are generated by neurons in the suprachiasmatic nuclei (SCN) of the hypothalamus. To better understand whether intrinsic properties of SCN neurons differ in anophthalmic and sighted mice, we examined the electrical activity of these neurons in slices, using single unit recordings, ionophoresis and bath perfusion of agonists and antagonists of known SCN neurotransmitters. Lucifer yellow was injected to characterize morphology. In controls, in daytime, units fired at a higher rate (44% at ≥5 Hz) than at night (21% at ≥5 Hz) and with regular interspike intervals versus irregular intervals nocturnally. In anophthalmics four firing patterns were observed as follows: (1) irregular at <5 spikes/s (70% of the total); (2) regular at ≥5 spikes/s; (<10%); (3) irregular bursts (20%); (4) regular bursts (<1%). Most neurons were inhibited by GABA, but a few were excited in controls. Blocking synaptic transmission with low Ca/high Mg increased the frequency and regularized the pattern of previously irregular discharges both in anophthalmics and controls. Bicuculline (10 M), a GABA A antagonist, had a similar effect. These data suggest that the characteristic irregular firing pattern of anophthalmics, and of controls at night, results from extrinsic, at least in part, GABAergic input.

ABSTRACT

The immunomodulator, imiquimod, has been shown to have antiviral and antitumor properties in animal models. It also has been reported to alter cytokine levels in both animals and humans. However, because imiquimod appeared to be emetic, studies were conducted to determine the degree of sensitivity, and the pathways involved. Subcutaneous administration of ≥10 mg/kg imiquimod to ferrets elicited emesis with latencies as short as 2′; 12 and 15 mg/kg were optimal doses. Emetic responsiveness was eliminated by complete ablation of the area postrema, but was unaffected by bilateral supradiaphragmatic section of the vagus nerve. This indicates that the emesis is produced by an activation of the chemoreceptor trigger zone B the area postrema. Ferret brain stem slices (450 μm) were preincubated in oxygenated Krebs-Ringer and then mounted in a submerged slice recording chamber. Extracellular recordings of spontaneous and ionophoretically evoked activity of area postrema neurons were obtained for up to 8 h, while the effect of bath-applied imiquimod was determined. Under control conditions, neurons showed a low frequency spontaneous discharge. Introduction of imiquimod (concentration range, 1 × 10−7to 5 × 10−8M) had no effect on neuronal firing. With ionophoresis of glutamate from an independent micropipette, a brief excitatory response was obtained. We conclude that imiquimod does not directly excite area postrema neurons. It is likely that imiquimod causes synthesis and release of some unknown emetic substance(s), very possibly cytokines.

Brief transient ischemia causes a delayed neuronal death of pyramidal neurons in the CA1 area of hippocampus after a period of hyperexcitability. We have previously shown that the hyperexcitability is due to an increase in an N-methyl-d-aspartate (NMDA) component of the response. In the present study, we recorded intracellularly from pyramidal neurons in CA1 and find that there is little change in membrane potential or input resistance at this point in time. The dramatic increase in the NMDA component of the synaptic response is a result of a significant reduction in the ability of Mg2+to induce a normal voltage-dependent blockade of the response. In spite of the relatively normal membrane properties, there is at this time a significant reduction in the amplitude of the population excitatory potential and a near total loss of long-term potentiation. In contrast, post-tetanic potentiation is unchanged in magnitude and character. These observations suggest more severe damage to the neuron than indicated by the membrane potential and resistance. When single neurons were injected with horseradish peroxidase and visualized after the electrophysiological recording, we found extensive beading of the dendrites in both the apical and basal regions, presumably reflecting a disproportionate damage to the dendritic areas, which are the primary sites of the excitatory amino acid synapses onto the neuron. These observations are consistent with the hypothesis that transient ischemia causes a fundamental change in the NMDA-activated ion channel such that Mg2+is no longer able to block the response, resulting in increased entry of calcium into synaptic regions, which causes dendritic damage that progresses to neuronal cell death.

Repeatedly it was reported that a short ischemic episode may ameliorate biochemical and morphological impairment upon succeeding severe ischemia. We investigated whether the pattern of respiratory enzyme activity (RA), adeninenucleotides, and membrane potential in hippocampal slices following low-dosein vivo(20 mg/kg) and high-dosein vitro(1 mM) application of 3-nitropropionic acid (3-np), a specific inhibitor of succinic dehydrogenase (SDH), indicates a similar tolerance phenomenon. One hourin vivotreatment decreased RA, spectrophotometrically quantitated by intensity of staining with 2,3,5-triphenyltetrazolium chloride (TTC), to 48 ± 5% (mean±SE;P<0.01). Intermittent increase after 2 h (79±5%;P<0.05) was followed by gradual decline to 48 ± 16% (P<0.01) after 8 h. The intermittent increase predominated in stratum pyramidale of hippocampal region CA1 (CA1SP) vs CA3 (CA3SP) (89±6% vs 57±6% of control;P<0.01). ATP levels paralleled the intensity of average (CA1SP, CA3SP, plus CA1 stratum radiatum) TTC staining (r=0.93). After pretreatment with 3-npin vivofor 1 h, no further decrease of RA upon 30-minin vitrotreatment was seen in any region. At all other times, RA declined further uponin vitrotreatment (P<0.01). Compared to 1-hin vivotreatment, hyperpolarization of CA1SPpyramidal cells uponin vitroapplication of 1 mM3-np was reduced after 8-h pretreatmentin vivo(P<0.04). At this time, depolarization upon glibenclamide (10 μM), an antagonist at KATP-channels, was reduced. We conclude that the severity of impairment of oxidative phosphorylation upon repeated inhibition of SDHin vivoandin vitrois not increased in an additive manner. At appropriate times, relative protection against further decrease of energy metabolism is observed—chemical preconditioning. Activation of KATP-channels is associated with chemical preconditioning.

Neuronal cell death during impaired energy metabolism is often attributed to increased activity at glutamate receptors, but this increase has not been directly demonstrated. We recorded responses to glutamate and N-methyl-d-aspartate in hippocampal slice CA1 neurons and glia while inhibiting mitochondrial complex II with 3-nitropropionic acid. As the period of inhibition increased, neuronal depolarization following bath application of glutamate (5 mM) or N-methyl-d-aspartate (50 μM) increased dramatically. However, depolarization upon iontophoresis of glutamate and N-methyl-d-aspartate decreased with time. A transient hyperpolarization, reflecting electrogenic sodium pump activity, was present immediately after responses to iontophoretic glutamate agonists. In the presence of the inhibitor, this hyperpolarization decreased and eventually disappeared. Even the repolarization seen upon washing of the iontophoretic or bath application of glutamate or N-methyl-d-aspartate was incomplete. Glial depolarization upon bath application of glutamate increased during inhibition, while glial depolarization upon application of N-methyl-d-aspartate decreased. Application of the N-methyl-d-aspartate antagonists aminophosphonovaleric acid (100 μM) or MK-801 (20 μM) resulted in a delay of further depolarization when applied early during the terminal decay of membrane potential following metabolic inhibition. We conclude that during impaired oxidative phosphorylation the failure of repolarizing mechanisms, not potentiated neuronal depolarization by excitants, is the primary cause of the terminal depolarization. Large glial depolarization increases the demand for neuronal ion exchange which cannot be met in situations of reduced energy metabolism.

The involvement of protein kinase C in long-term potentiation was investigated in the mossy fiber-CA3 pathway in anin vitro slice preparation of rat hippocampus. Tetanic stimulation induced stable long-term potentiation in the mossy fiber-CA3 pathway which was not affected byN-methyl-d-aspartate receptor antagonists. Long-term potentiation was not induced in the presence of a protein kinase C inhibitor, sphingosine. Application of 1 μM phorbol-12,13-diacetate, an activator of protein kinase C, potentiated the synaptic response by about 400% and this potentiation was completely reversible upon washing. Sphingosine blocked the potentiation when it was applied before protein kinase C activation by phorbol-12,13-diacetate. However, sphingosine had no effect on the potentiation when it was applied after the synaptic response was potentiated to a plateau following phorbol-12,13-diacetate perfusion. Long-term potentiation and phorbol ester-induced potentiation were not additive when phorbol-12,13-diacetate was applied after induction of long-term potentiation, suggesting that long-term potentiation and phorbol-12,13-diacetate activate the same protein kinase C pool. The enhanced response caused by phorbol-12,13-diacetate returned to the long-term potentiation level after wash-out of phorbol-12,13-diacetate. Thus the cellular changes underlying long-term potentiation are long-lasting or permanent, while those caused by phorbol-12,13-diacetate are not. However, if tetanic stimulation was induced during prolonged phorbol-12,13-diacetate application (1 h), a potentiation similar in amplitude to long-term potentiation was induced but the population response returned to the control pre-long-term potentiation level after 2 h of washing. The potentiation following tetanic stimulation during prolonged application of phorbol-12,13-diacetate was blocked in the presence ofd-2-amino-5-phosphonovaleric acid, aN-methyl-d-aspartate receptor antagonist. Thus, in the presence of phorbol esters theN-methyl-d-aspartate-independent long-term potentiation is occluded but a transient potentiation appears, presumably due to hyperexcitability and activation ofN-methyl-d-aspartate receptors in recurrent pathways of area CA3. NormalN-methyl-d-asparate-independent long-term potentiation could be induced after the 2 h washout period and now was maintained. In conclusion, protein kinase C activation is essential but not sufficient for long-term potentiation in the mossy fiber-CA3 pathway and when stimulated by application of phorbol esters produces a large and reversible synaptic potentiation.