Calcium regulation of a slow post-spike hyperpolarization in vagal afferent neurons

Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPslow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHPslow in nodose neurons displays a slow rise time to peak (0.3–0.5 s) and a long duration (3–15 s). The slow kinetics of the AHPslow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ influx via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPslow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPslow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency adaptation in vagal afferent neurons.

Ensemble codes involving hippocampal neurons are at risk during delayed performance tests

Multielectrode recording techniques were used to record ensemble activity from 10 to 16 simultaneously active CA1 and CA3 neurons in the rat hippocampus during performance of a spatial delayed-nonmatch-to-sample task. Extracted sources of variance were used to assess the nature of two different types of errors that accounted for 30% of total trials. The two types of errors included ensemble “miscodes” of sample phase information and errors associated with delay-dependent corruption or disappearance of sample information at the time of the nonmatch response. Statistical assessment of trial sequences and associated “strength” of hippocampal ensemble codes revealed that miscoded error trials always followed delay-dependent error trials in which encoding was “weak,” indicating that the two types of errors were “linked.” It was determined that the occurrence of weakly encoded, delay-dependent error trials initiated an ensemble encoding “strategy” that increased the chances of being correct on the next trial and avoided the occurrence of further delay-dependent errors. Unexpectedly, the strategy involved “strongly” encoding response position information from the prior (delay-dependent) error trial and carrying it forward to the sample phase of the next trial. This produced a miscode type error on trials in which the “carried over” information obliterated encoding of the sample phase response on the next trial. Application of this strategy, irrespective of outcome, was sufficient to reorient the animal to the proper between trial sequence of response contingencies (nonmatch-to-sample) and boost performance to 73% correct on subsequent trials. The capacity for ensemble analyses of strength of information encoding combined with statistical assessment of trial sequences therefore provided unique insight into the “dynamic” nature of the role hippocampus plays in delay type memory tasks.

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Δ272–299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.

Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.

Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.