Somatic Region Research Articles

Effect of presynaptic membrane potential on electrical vs. chemical synaptic transmission

The growing realization that electrical coupling is present in the mammalian brain has sparked renewed interest in determining its functional significance and contrasting it with chemical transmission. One question of interest is whether the two types of transmission can be selectively regulated, e.g., if a cell makes both types of connections can electrical transmission occur in the absence of chemical transmission? We explore this issue in an experimentally advantageous preparation. B21, the neuron we study, is an Aplysia sensory neuron involved in feeding that makes electrical and chemical connections with other identified cells. Previously we demonstrated that chemical synaptic transmission is membrane potential dependent. It occurs when B21 is centrally depolarized prior to and during peripheral activation, but does not occur if B21 is peripherally activated at its resting membrane potential. In this article we study effects of membrane potential on electrical transmission. We demonstrate that maximal potentiation occurs in different voltage ranges for the two types of transmission, with potentiation of electrical transmission occurring at more hyperpolarized potentials (i.e., requiring less central depolarization). Furthermore, we describe a physiologically relevant type of stimulus that induces both spiking and an envelope of depolarization in the somatic region of B21. This depolarization does not induce functional chemical synaptic transmission but is comparable to the depolarization needed to maximally potentiate electrical transmission. In this study we therefore characterize a situation in which electrical and chemical transmission can be selectively controlled by membrane potential.

Extracellular DC electric fields induce nonuniform membrane polarization in rat hippocampal CA1 pyramidal neurons

Non-synaptic interactions among neurons via extracellular electric fields may play functional roles in the CNS. Previously in a study using voltage-sensitive dye imaging, we reported characteristic membrane polarization profiles in the CA1 region of hippocampal slices during exposure to extracellular DC fields: slow monophasic polarization in somatic region and biphasic polarization (fast polarization and following slow repolarization) in mid-dendritic region. Here, using optical imaging and patch-clamp recordings, we showed that CA1 pyramidal neurons indeed show the characteristic polarization in response to DC fields, and investigated the mechanism underlying the profiles. Both the monophasic and biphasic polarization could be fitted with a double exponential function. The τs (ms) were 12.6±2.5 and 56.0±4.7 for the monophasic polarization, and 14.2±1.2 and 42.2±2.8 for the biphasic polarization. Based on our previous theoretical studies, we hypothesized that lower resistivity in the distal apical dendrites is responsible for generating the characteristic polarization profiles. We tested this hypothesis by removing the distal apical dendrites or by blocking ion channel-mediated conductance. Removal of distal dendrites caused drastic changes in the polarization profiles, e.g. biphasic polarization was damped. However, none of the blockers tested had a marked effect on the biphasic polarization. Our results demonstrate the importance of the apical dendrite for generating the characteristic polarization profiles, and suggest that voltage-activated conductance, including HCN channel-mediated conductance, had only minor contributions to these profiles. These findings provide a better understanding of how neurons in the CNS respond to extracellular electric fields.

Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

Impaired striatal GABA transmission in experimental autoimmune encephalomyelitis

Synaptic dysfunction triggers neuronal damage in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). While excessive glutamate signaling has been reported in the striatum of EAE, it is still uncertain whether GABA synapses are altered. Electrophysiological recordings showed a reduction of spontaneous GABAergic synaptic currents (sIPSCs) recorded from striatal projection neurons of mice with MOG (35−55)-induced EAE. GABAergic sIPSC deficits started in the acute phase of the disease (20–25 days post immunization, dpi), and were exacerbated at later time-points (35, 50, 70 and 90 dpi). Of note, in slices they were independent of microglial activation and of release of TNF-α. Indeed, sIPSC inhibition likely involved synaptic inputs arising from GABAergic interneurons, because EAE preferentially reduced sIPSCs of high amplitude, and was associated with a selective loss of striatal parvalbumin (PV)-positive GABAergic interneurons, which contact striatal projection neurons in their somatic region, giving rise to more efficient synaptic inhibition. Furthermore, we found also that the chronic persistence of pro-inflammatory cytokines were able, per se, to produce profound alterations of electrophysiological network properties, that were reverted by GABA administration. The results of the present investigation indicate defective GABA transmission in MS models depending from alteration of PV cells number and, in part, deriving from the effects of a chronic inflammation, and suggest that pharmacological agents potentiating GABA signaling might be considered to limit neuronal damage in MS patients.

Different sized somatic NF1 locus rearrangements in neurofibromatosis 1-associated malignant peripheral nerve sheath tumors

Neurofibromatosis type1 (NF1) patients are at increased risk of developing both benign (neurofibromas) and malignant (malignant peripheral nerve sheath tumors, MPNST) tumors. Molecular data on tumor progression are scarce, and few studies have compared the NF1 locus copy number in these two tumor types. To further explore the role of such NF1 locus rearrangements in NF1 tumorigenesis, and the likely disruption to the associated genes, the NF1 gene region was analyzed in NF1-associated tumors. DNA from three MPNSTs and one neurofibroma, excised from three unrelated NF1 patients, were analyzed using an NF1 region customized array-based comparative genomic hybridization. The somatic NF1 inactivation mutational mechanisms associated with MPNSTs appear to be different from those in benign neurofibromas. Interestingly, the MPNST-associated deletion breakpoints did not involve the paralogous repetitive sequences that are involved in most germline NF1 deletions. The somatic smallest common region of deletion overlap, however, was restricted to approximately the same ~2.2-Mb interval that encompassed most of the genes deleted in NF1 recurrent constitutional deletions. A number of genes in addition to NF1 on 17q (centromere to 17q24.2) may be involved in MPNST development. A larger study is warranted to confirm these findings. As NF1 patients with such germline NF1 deletions do exhibit increased risk of developing MPNST, these present findings emphasize the likely role of at least some of these NF1 flanking genes in MPNST pathobiology.

The Mammalian Interaural Time Difference Detection Circuit Is Differentially Controlled by GABAB Receptors during Development

Throughout development GABA(B) receptors (GABA(B)Rs) are widely expressed in the mammalian brain. In mature auditory brainstem neurons, GABA(B)Rs are involved in the short-term regulation of the strength and dynamics of excitatory and inhibitory inputs, thus modulating sound analysis. During development, GABA(B)Rs also contribute to long-term changes in input strength. Using a combination of whole-cell patch-clamp recordings in acute brain slices and immunostainings in gerbils, we characterized developmental changes in GABA(B)R-mediated regulation of synaptic inputs to neurons in the medial superior olive (MSO), an auditory brainstem nucleus that analyzes interaural time differences (ITDs). Here, we show that, before hearing onset, GABA(B)R-mediated depression of transmitter release is much stronger for excitation than inhibition, whereas in mature animals GABA(B)Rs mainly control the inhibition. During the same developmental period, GABA(B)R immunoreactivity shifts from the dendritic to the somatic region of the MSO. Furthermore, only before hearing onset (postnatal day 12), stimulation of the fibers originating in the medial and the lateral nucleus of the trapezoid body (MNTB and LNTB) activates GABA(B)Rs on both the inhibitory and the excitatory inputs. After hearing onset, GAD65-positive endings devoid of glycine transporter reactivity suggest GABA release from sources other than the MNTB and LNTB. At this age, pharmacological increase of spontaneous synaptic release activates GABA(B)Rs only on the inhibitory inputs. This indicates not only a profound inhibitory effect of GABA(B)Rs on the major inputs to MSO neurons in neonatal animals but also a direct modulatory role of GABA(B)Rs for ITD analysis in the MSO of adult animals.

Mitochondrial mutations in hepatocellular carcinomas and fibrolamellar carcinomas

Mitochondrial mutations are well documented in hepatocellular carcinoma, but their role in carcinogenesis remains unclear. To clarify their significance, a comprehensive analysis was performed of hepatocellular carcinomas (N=24), including quantifying the total mitochondrial DNA levels, quantifying the levels of mitochondrial DNA with the common deletion, and complete sequencing of the mitochondrial control region. In addition, these studies were expanded and reinforced by analysis of fibrolamellar carcinomas (N=15), a unique type of liver carcinoma that has increased numbers of mitochondria on electron microscopy. Overall, approximately 50% of hepatocellular carcinomas had lower levels of total mitochondrial DNA than paired non-neoplastic tissues. Interestingly, despite their increased numbers of mitochondria, primary fibrolamellar carcinomas had lower levels of total mitochondrial DNA. In contrast, metastatic fibrolamellar carcinomas had greatly increased mitochondrial DNA levels. Overall, deletions in the control region were associated with lower total DNA levels in typical hepatocellular carcinoma, but somatic single base pair mutations were not. In fact, almost all single base pair mutations were either reversions to the wild-type sequence or known population polymorphisms, strongly suggesting they are not directly oncogenic. Complete sequencing of the entire mitochondrial genome in fibrolamellar carcinomas identified several somatic mutations, but no consistent pattern of mutations was found. Overall, the levels of the common deletion were highest in tissues with lower total mitochondrial DNA. In conclusion, control region deletions, but not somatic mutations, may influence total DNA copy numbers. Somatic control region mutations in hepatocellular carcinoma are not directly oncogenic but instead may be adaptive.

Retrograde analysis of the cerebellar projections to the posteroventral part of the ventral lateral thalamic nucleus in the macaque monkey

The organization of cerebellothalamic projections was investigated in macaque monkeys using injections of retrograde tracers (cholera toxin B and fluorescent dextrans) in the posteroventral part of the ventrolateral thalamic nucleus (VLpv), the main source of thalamic inputs to the primary motor cortex. Injections that filled all of VLpv labeled abundant neurons that were inhomogeneously distributed among many unlabeled cells in the deep cerebellar nuclei (DCbN). Single large pressure injections made in face-, forelimb-, or hindlimb-related portions of VLpv using physiological guidance labeled numerous neurons that were broadly dispersed within a coarse somatotopographic anteroposterior (foot to face) gradient in the dentate and interposed nuclei. Small iontophoretic injections labeled fewer neurons with the same somatotopographic gradient, but strikingly, the labeled neurons in these cases were as broadly dispersed as in cases with large injections. Simultaneous injections of multiple tracers in VLpv (one tracer per somatic region with no overlap between injections) confirmed the general somatotopography but also demonstrated clearly the overlapping distributions and the close intermingling of neurons labeled with different tracers. Significantly, very few neurons (<2%) were double-labeled. This organizational pattern contrasts with the concept of a segregated "point-to-point" somatotopy and instead resembles the complex patterns that have been observed throughout the motor pathway. These data support the idea that muscle synergies are represented anatomically in the DCbN by a general somatotopography in which intermingled neurons and dispersed but selective connections provide the basis for plastic, adaptable movement coordination of different parts of the body. Indexing terms:

77 Somatic mtDNA control region mutations and their effects in down syndrome with Alzheimer dementia

77 Somatic mtDNA control region mutations and their effects in down syndrome with Alzheimer dementia

Mechanoafferent Neuron With An Inexcitable Somatic Region: Consequences for the Regulation of Spike Propagation and Afferent Transmission

In the Aplysia mechanoafferent B21, afferent transmission is in part regulated via the control of active spike propagation. When B21 is peripherally activated at its resting membrane potential, spikes fail to propagate to an output process, and afferent transmission does not occur. In this report, we show that the propagation failure is in part a result of the fact that the somatic region of B21 is relatively inexcitable. We isolate this region and demonstrate that net currents evoked by depolarizing pulses are outward. Furthermore, we show that all-or-none spikes are not triggered when current is injected. Previous reports have, however shown that spiking is triggered when current is somatically injected and cells are intact. We demonstrate that spikes evoked under these circumstances do not originate in the soma. Instead they originate in an adjacent part of the neuron that is excitable (the medial process). In summary, we show that the mechanoafferent B21 consists of excitable input and output processes separated by a relatively inexcitable somatic region. A potential advantage of this arrangement is that somatic depolarization can be used to modify spike propagation from the input to the output processes without altering the encoding of peripherally generated activity.

Modulation of calcium wave propagation in the dendrites and to the soma of rat hippocampal pyramidal neurons.

Repetitive synaptic stimulation in the stratum radiatum (SR) evokes large amplitude Ca2+ waves in the thick apical dendrites of hippocampal CA1 pyramidal neurons. These waves are initiated by activation of metabotropic glutamate receptors (mGluRs), which mobilize inositol-1,4,5-trisphospate (IP3) and release Ca2+ from intracellular stores. We explored mechanisms that modulate the spatial properties of these waves. Higher stimulus current evoked waves of increasing spatial extent. Most waves did not propagate through the soma; the majority stopped close to the junction of the soma and apical dendrite. Pairing strong stimulation with one electrode and subthreshold stimulation with another (associative activation) extended the waves distally but failed to extend waves into the cell body. Pairing synaptic stimulation with backpropagating action potentials enhanced the likelihood of wave generation but did not extend the waves to the somatic region. Priming the stores with Ca2+ entry through voltage dependent channels modulated wave properties but did not extend them past the dendrites. These results are consistent with propagation failing due to the dilution of synaptically generated IP3 as it diffuses into the large volume of the soma (impedance mismatch). Synaptically activating waves in the presence of low concentrations of carbachol, which probably increased the tonic level of IP3 throughout the cell, enhanced the extent of propagation and generated waves that invaded the soma, as long as low-affinity indicators were used to detect the [Ca2+]i changes. Consistent with this explanation direct injection of IP3 into the soma promoted wave propagation into this region. Ca2+ waves that propagated through the cell body were interesting because they did not fill the volume of the soma, but passed through the centre, often with large amplitude. These waves may be particularly effective in activating gene expression and protein synthesis.

L-type Ca2+ channels contribute to current-evoked spike firing and associated Ca2+ signals in cerebellar Purkinje neurons

The physiological properties of Purkinje neurons play a central role in their ability to regulate information transfer through the cerebellum. A number of ion channels contribute to Purkinje neuron physiology including an abundance of P-type Ca2+ channels, particularly in the dendritic region. Purkinje neurons also express L-type Ca2+ channels both during development and in the mature state. However, a role for L-type channels in Purkinje neuron physiology has yet to be fully defined. In the current study we used physiological recordings from cultured Purkinje neurons and the L-type Ca2+ channel agonist S-(-)-Bay K to assess a potential role for L-type Ca2+ channels in spike firing. Results show that Bay K alters current-evoked spike firing in young, immature Purkinje neurons without dendritic structure and in older, more mature Purkinje neurons with dendritic structure. Bay K also enhanced Ca2+ signals associated with the current-evoked spike firing. The effect of Bay K was more prominent in the young Purkinje neurons than in the older Purkinje neurons, suggesting that L-type Ca2+ channels may be more important in the Purkinje neuron physiology during the early stages of development rather than at mature stages. In the older Purkinje neurons, immunohistochemical studies using antibodies to L-type Ca2+ channels showed more intense immunolabeling in the somatic region than in the dendritic region. This result suggests that L-type Ca2+ channels may play a more important role in somatic physiology than dendritic physiology, whereas P-type channels may play a more important role in dendritic physiology.

Recording, analysis, and function of dendritic voltage-gated channels

Ever since the publication of the Hamill et al. [Hamill et al., Pflügers Arch, 391:85-100, 1981] paper and the following increase in popularity of acute brain slice preparations, there has been a large increase in the volume of publications investigating voltage-gated channels in the central nervous system using the patch-clamp technique. In the preceding decade, investigations of voltage-gated channels have moved out of the somatic region into dendrites providing much needed information about dendritic voltage-gated channels. In this study, we review some aspects related to the investigation of voltage-gated ion channels in dendrites: recording, analysis, and function.

Mitochondrial Genome Instability in AML Is Associated with Reduction in the Mitochondrial Replication.

Genetic changes in mitochondrial DNA (mtDNA) have been hypothesized more widely to play important roles in senescence, autoimmune disease and malignancy because of a paucity of introns and limited repair mechanisms. Malfunction of mismatch repair genes produces genome instability which plays an important role in the development of human cancers. The mtDNA markers for mitochondrial genome instability (mtGI) were point mutations, insertions, deletions and length changes in homopolymeric nucleotide tracts. We investigated the mtGI in AML cells and its effect to alteration in mtDNA copy number. Forty-eight matched AML bone marrow and buccal mucosa samples, and blood samples from 57 control subjects were collected after receiving Institutional Review Board approval and informed consent. We directly sequenced the control region, the tRNA leucine 1 gene plus a part of NADH dehydrogenase (ND)1 and cytochrome b (CYTB) of mtDNA. In an attempt to investigate mtGI, we carried out a qualitative and quantitative profiling mtDNA length heteroplasmies of six mtGIs (np 303–315 poly C, np 16184–16193 poly C, np 514–511 CA repeats, np 3566–3572 poly C, np 12385–12391 poly C and np 12418–12426 poly A) using a size-based PCR product separation by capillary electrophoresis (ABI Prism Genotyper version 3.1). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen). In the current study, we detected a large number of polymorphisms as well as new mtDNA variants. A total of 606 mtDNA sequence variants were identified. Among these, 15 mtDNA variants were identified as novel mutations that were absent from corresponding buccal mucosa, control subjects and established mtDNA polymorphism databases. In the control region, we found two types of mtDNA alterations - base substitutions and small deletions/insertions as well as the length heteroplasmies in the np 303 to 315 poly-C, np 16184 to 16193 poly-C and 514–515 CA repeats. Seven patients (15%) had leukemia cell-specific mtDNA substitution mutations in the ND1 and CYTB genes. Somatic mtDNA control region mutations found in this study preferentially altered known mtDNA regulatory elements. AML cells had about a two-fold decrease in mtDNA copy number compared with the results from control subjects (63 x 106 molecules/ul ± 23 x 106 vs 122 x 106 molecules/ul ± 73 x 106). Our results are consistent with a recent observation that carcinogenesis in the liver, kidney and lung involves a decrease of the cellular mitochondrial content and decreased mtDNA copy number (Mutat Res 2004; 547:71–78). In conclusion, mtGI including point mutations, length changes (insertions or deletions) in homopolymeric tracts commonly occurred in AML cells and reduction in mtDNA copy number may result from either mtDNA control region mutations or impairment of mitochondrial biogenesis. These findings suggest that biogenesis of mitochondria is repressed in the leukemogenesis process of the human hematopoietic tissue.

Proximal Persistent Na+Channels Drive Spike Afterdepolarizations and Associated Bursting in Adult CA1 Pyramidal Cells

In many principal brain neurons, the fast, all-or-none Na+ spike initiated at the proximal axon is followed by a slow, graded after depolarization (ADP). The spike ADP is critically important in determining the firing mode of many neurons; large ADPs cause neurons to fire bursts of spikes rather than solitary spikes. Nonetheless, not much is known about how and where spike ADPs are initiated. We addressed these questions in adult CA1 pyramidal cells, which manifest conspicuous somatic spike ADPs and an associated propensity for bursting, using sharp and patch microelectrode recordings in acutely isolated hippocampal slices and single neurons. Voltage-clamp commands mimicking spike waveforms evoked transient Na+ spike currents that declined quickly after the spike but were followed by substantial sustained Na+ spike after currents. Drugs that blocked the persistent Na+ current (INaP), markedly suppressed the sustained Na+ spike after currents, as well as spike ADPs and associated bursting. Ca2+ spike after currents were much smaller, and reducing them had no noticeable effect on the spike ADPs. Truncating the apical dendrites affected neither spike ADPs nor the firing modes of these neurons. Application of INaP blockers to truncated neurons, or their focal application to the somatic region of intact neurons, suppressed spike ADPs and associated bursting, whereas their focal application to distal dendrites did not. We conclude that the somatic spike ADPs are generated predominantly by persistent Na+ channels located at or near the soma. Through this action, proximal INaP critically determines the firing mode and spike output of adult CA1 pyramidal cells.

Unilateral cortical application of tumor necrosis factor α induces asymmetry in Fos- and interleukin-1β-immunoreactive cells within the corticothalamic projection

A unilateral microinjection of tumor necrosis factor alpha (TNFα) (150 ng) onto the primary somatosensory cortex induces state-dependent asymmetries in electroencephalographic (EEG) slow wave activity during non-rapid eye movement sleep in rats [H. Yoshida, Z. Peterfi, F. Garcia-Garcia, R. Kirkpatrick, T. Yasuda, J.M. Krueger, State-specific asymmetries in EEG slow wave activity induced by local application of TNF alpha, Brain Res. 1009 (2004) 129–136]. In the current study, analogous TNFα injections were performed to determine Fos- and interleukin-1β (IL1β) immunoreactivity (IR). A unilateral microinjection of TNFα increased the number of Fos- and IL1β-IR cells in the primary somatosensory cortex relative to the contralateral side that received heat-inactivated TNFα. These asymmetric TNFα-induced increases in the number of Fos- and IL1β-IR cells were evident along the outside surface of the cortex (mainly layers II and III) in a restricted rostral to caudal zone. Asymmetrical increases in the number of Fos-IR cells were also observed in the subcortical region that receives the main cortical projection from the somatosensory cortex, the somatic region of the reticular nucleus of the thalamus (reticular thalamus). The IL1β-IR cells double-labeled with glial fibrillary acidic protein (GFAP), suggesting that many of the IL1β-IR cells were astrocytes. The number of the IL1β-IR cells in the reticular thalamus increased significantly ipsilateral to the TNFα injection. Current results indicated that Fos- and IL1β-IR may be utilized to study the functional neuroanatomy involved in the TNFα-mediated state-dependent enhancement of EEG slow wave activity.

A transitional period of Ca2+‐dependent spike afterdepolarization and bursting in developing rat CA1 pyramidal cells

During postnatal development neurones display discharge behaviours that are not present in the adult, yet they are essential for the normal maturation of the nervous system. Neonatal CA1 pyramidal cells, like their adult counterparts, fire regularly, but excitatory GABAergic transmission drives them to generate spontaneous high-frequency bursts until postnatal day (P) 15. Using intracellular recordings in hippocampal slices from rats at P8 to P25, we show herein that as the network-driven burst activity fades out, most CA1 pyramidal cells become intrinsically bursting neurones. The incidence of intrinsic bursters begins to rise at P11 and attains a peak of 74% by P18-P19, after which it decreases over the course of a week, disappearing almost entirely at P25. Analysis of the effects of different voltage-gated Ca2+ and Na+ channel antagonists, applied focally to proximal and distal parts of developing neurones, revealed a complex burst mechanism. Intrinsic bursting in developing neurones results from 'ping-pong' interplay between a back-propagating spike that activates T/R- and L-type voltage-gated Ca2+)channels in the distal apical dendrites and persistent voltage-gated Na+ channels in the somatic region. Thus, developing pyramidal neurones transitionally express not only distinctive synaptic properties, but also unique intrinsic firing patterns, that may contribute to the ongoing formation and refinement of synaptic connections.

Spike train dynamics predicts theta-related phase precession in hippocampal pyramidal cells.

According to the temporal coding hypothesis, neurons encode information by the exact timing of spikes. An example of temporal coding is the hippocampal phase precession phenomenon, in which the timing of pyramidal cell spikes relative to the theta rhythm shows a unidirectional forward precession during spatial behaviour. Here we show that phase precession occurs in both spatial and non-spatial behaviours. We found that spike phase correlated with instantaneous discharge rate, and processed unidirectionally at high rates, regardless of behaviour. The spatial phase precession phenomenon is therefore a manifestation of a more fundamental principle governing the timing of pyramidal cell discharge. We suggest that intrinsic properties of pyramidal cells have a key role in determining spike times, and that the interplay between the magnitude of dendritic excitation and rhythmic inhibition of the somatic region is responsible for the phase assignment of spikes.

Chronic Ethanol Treatment and Withdrawal Alter ACPD‐Evoked Calcium Signals in Developing Purkinje Neurons

Alcohol exposure during human fetal development can result in fetal alcohol syndrome, a condition characterized by central nervous system dysfunction. Detailed studies in animal models of fetal alcohol syndrome show that the cerebellar region is particularly sensitive to alcohol exposure during early development; however, the cellular mechanisms underlying the alcohol sensitivity of the immature cerebellum are poorly understood. Primary neuronal cultures of cerebellar cells were prepared from embryonic day 20 rat pups. Cultures were exposed to ethanol (33 mM; 150 mg/100 ml) during the main period of morphological development of the Purkinje neurons, from 6 to 17 days in vitro. After the ethanol treatment, the response of Purkinje neurons to the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 microM) was examined in parallel fura-2 Ca2+ imaging and current-clamp experiments. In an additional set of experiments, ethanol-treated cultures were allowed to withdraw from ethanol overnight before recordings were performed. In Ca2+ imaging studies, the mean peak amplitude of ACPD-evoked Ca2+ signals was depressed in the dendritic region of chronic ethanol-treated Purkinje neurons compared with control neurons (p < 0.05, unpaired t test), whereas there was no apparent difference in the somatic region. In contrast, peak ACPD-evoked Ca2+ signals were enhanced in both the somatic and dendritic regions of withdrawn Purkinje neurons compared with control neurons. Parallel current-clamp studies showed no consistent effect of chronic ethanol treatment or ethanol withdrawal on the membrane response to ACPD. These results show that prolonged ethanol exposure and early withdrawal lead to alterations in mGluR-evoked Ca2+ signaling in cerebellar Purkinje neurons. Metabotropic GluRs in the Purkinje neuron play important roles in cerebellar development and function, suggesting that alterations of mGluR signaling pathways by ethanol may play a key role in the actions of ethanol on the developing cerebellum.

Chronic Ethanol Treatment and Withdrawal Alter ACPD-Evoked Calcium Signals in Developing Purkinje Neurons

Background: Alcohol exposure during human fetal development can result in fetal alcohol syndrome, a condition characterized by central nervous system dysfunction. Detailed studies in animal models of fetal alcohol syndrome show that the cerebellar region is particularly sensitive to alcohol exposure during early development; however, the cellular mechanisms underlying the alcohol sensitivity of the immature cerebellum are poorly understood. Methods: Primary neuronal cultures of cerebellar cells were prepared from embryonic day 20 rat pups. Cultures were exposed to ethanol (33 mM; 150 mg/100 ml) during the main period of morphological development of the Purkinje neurons, from 6 to 17 days in vitro. After the ethanol treatment, the response of Purkinje neurons to the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 μM) was examined in parallel fura-2 Ca2+ imaging and current-clamp experiments. In an additional set of experiments, ethanol-treated cultures were allowed to withdraw from ethanol overnight before recordings were performed. Results: In Ca2+ imaging studies, the mean peak amplitude of ACPD-evoked Ca2+ signals was depressed in the dendritic region of chronic ethanol–treated Purkinje neurons compared with control neurons (p < 0.05, unpaired t test), whereas there was no apparent difference in the somatic region. In contrast, peak ACPD-evoked Ca2+ signals were enhanced in both the somatic and dendritic regions of withdrawn Purkinje neurons compared with control neurons. Parallel current-clamp studies showed no consistent effect of chronic ethanol treatment or ethanol withdrawal on the membrane response to ACPD. Conclusions: These results show that prolonged ethanol exposure and early withdrawal lead to alterations in mGluR-evoked Ca2+ signaling in cerebellar Purkinje neurons. Metabotropic GluRs in the Purkinje neuron play important roles in cerebellar development and function, suggesting that alterations of mGluR signaling pathways by ethanol may play a key role in the actions of ethanol on the developing cerebellum.