Domoic acid disrupts the activity and connectivity of neuronal networks in organotypic brain slice cultures.

Domoic acid is a neurotoxin produced by algae and is found in seafood during harmful algal blooms. As a glutamate agonist, domoic acid inappropriately stimulates excitatory activity in neurons. At high doses, this leads to seizures and brain lesions, but it is unclear how lower, asymptomatic exposures disrupt neuronal activity. Domoic acid has been detected in an increasing variety of species across a greater geographical range than ever before, making it critical to understand the potential health impacts of low-level exposure on vulnerable marine mammal and human populations. To determine whether prolonged domoic acid exposure altered neuronal activity in hippocampal networks, we used a custom-made 512 multi-electrode array with high spatial and temporal resolution to record extracellular potentials (spikes) in mouse organotypic brain slice cultures. We identified individual neurons based on spike waveform and location, and measured the activity and functional connectivity within the neuronal networks of brain slice cultures. Domoic acid exposure significantly altered neuronal spiking activity patterns, and increased functional connectivity within exposed cultures, in the absence of overt cellular or neuronal toxicity. While the overall spiking activity of neurons in domoic acid-exposed cultures was comparable to controls, exposed neurons spiked significantly more often in bursts. We also identified a subset of neurons that were electrophysiologically silenced in exposed cultures, and putatively identified those neurons as fast-spiking inhibitory neurons. These results provide evidence that domoic acid affects neuronal activity in the absence of cytotoxicity, and suggest that neurodevelopmental exposure to domoic acid may alter neurological function in the absence of clinical symptoms.

A novel cross-frequency coupling detection method using the generalized Morse wavelets.

BACKGROUND: Cross-frequency coupling (CFC) occurs when non-identical frequency components entrain one another. A ubiquitous example from neuroscience is low frequency phase to high frequency amplitude coupling in electrophysiological signals. Seminal work by Canolty revealed CFC in human ECoG data. Established methods band-pass the data into component frequencies then convert the band-passed signals into the analytic representation, from which we infer the instantaneous amplitude and phase of each component. Though powerful, such methods resolve signals with respect to time and frequency without addressing the multiresolution problem. NEW METHOD: We build upon the ground-breaking work of Canolty and others and derive a wavelet-based CFC detection algorithm that efficiently searches a range of frequencies using a sequence of filters with optimal trade-off between time and frequency resolution. We validate our method using simulated data and analyze CFC within and between the primary motor cortex and dorsal striatum of rats under ketamine-xylazine anesthesia. RESULTS: Our method detects the correct CFC in simulated data and reveals CFC between frequency bands that were previously shown to participate in corticostriatal effective connectivity. COMPARISON WITH EXISTING METHODS: Other CFC detection methods address the need to increase bandwidth when analyzing high frequency components but none to date permit rigorous bandwidth selection with no a priori knowledge of underlying CFC. Our method is thus particularly useful for exploratory studies. CONCLUSIONS: The method developed here permits rigorous and efficient exploration of a hypothesis space and is particularly useful when the frequencies participating in CFC are unknown.

Dense arrays of micro-needles for recording and electrical stimulation of neural activity in acute brain slices.

OBJECTIVE: This paper describes the design, microfabrication, electrical characterization and biological evaluation of a high-density micro-needle array. The array records from and electrically stimulates individual neurons simultaneously in acute slices of brain tissue. APPROACH: Acute slices, arguably the closest in-vitro model of the brain, have a damaged surface layer. Since electrophysiological recording methods rely heavily on electrode-cell proximity, this layer significantly attenuates the signal amplitude making the use of traditional planar electrodes unsuitable. To penetrate into the tissue, bypassing the tissue surface, and to record and stimulate neural activity in the healthy interior volume of the slice, an array of 61 micro-needles was fabricated. MAIN RESULTS: This device is shown to record extracellular action potentials from individual neurons in acute cortical slices with a signal to noise ratio of up to ∼15:1. Electrical stimulation of individual neurons is achieved with stimulation thresholds of 1.1-2.9 µA. SIGNIFICANCE: The novelty of this system is the combination of close needle spacing (60 µm), needle heights of up to 250 µm and small (5-10 µm diameter) electrodes allowing the recording of single unit activity. The array is coupled to a custom-designed readout system forming a powerful electrophysiological tool that permits two-way electrode-cell communication with populations of neurons in acute brain slices.

A statistical theory of long-term potentiation and depression.

The synaptic phenomena of long-term potentiation (LTP) and long-term depression (LTD) have been intensively studied for over twenty-five years. Although many diverse aspects of these forms of plasticity have been observed, no single theory has offered a unifying explanation for them. Here, a statistical "bin" model is proposed to account for a variety of features observed in LTP and LTD experiments performed with field potentials in mammalian cortical slices. It is hypothesized that long-term synaptic changes will be induced when statistically unlikely conjunctions of pre- and postsynaptic activity occur. This hypothesis implies that finite changes in synaptic strength will be proportional to information transmitted by conjunctions and that excitatory synapses will obey a Hebbian rule (Hebb, 1949). Using only one set of constants, the bin model offers an explanation as to why synaptic strength decreases in a decelerating manner during LTD induction (Mulkey & Malenka, 1992); why the induction protocols for LTP and LTD are asymmetric (Dudek & Bear, 1992; Mulkey & Malenka, 1992); why stimulation over a range of frequencies produces a frequency-response curve similar to that proposed by the BCM theory (Bienenstock, Cooper, & Munro, 1982; Dudek & Bear, 1992); and why this curve would shift as postsynaptic activity is changed (Kirkwood, Rioult, & Bear, 1996). In addition, the bin model offers an alternative to the BCM theory by predicting that changes in postsynaptic activity will produce vertical shifts in the curve rather than merely horizontal shifts.

Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches.

We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216-17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and beta-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k(cat) for the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant.

Prolonged synaptic integration in perirhinal cortical neurons.

Layer II/III of rat perirhinal cortex (PR) contains numerous late-spiking (LS) pyramidal neurons. When injected with a depolarizing current step, these LS cells typically delay spiking for one or more seconds from the onset of the current step and then sustain firing for the duration of the step. This pattern of delayed and sustained firing suggested a specific computational role for LS cells in temporal learning. This hypothesis predicts and requires that some layer II/III neurons should also exhibit delayed and sustained spiking in response to a train of excitatory synaptic inputs. Here we tested this prediction using visually guided, whole cell recordings from rat PR brain slices. Most LS cells (19 of 26) exhibited delayed spiking to synaptic stimulation (>1 s latency from the train onset), and the majority of these cells (13 of 19) also showed sustained firing that persisted for the duration of the synaptic train (5-10 s duration). Delayed and sustained firing in response to long synaptic trains has not been previously reported in vertebrate neurons. The data are consistent with our model that a circuit containing late spiking neurons can be used for encoding long time intervals during associative learning.

A new class of DNA glycosylase/apurinic/apyrimidinic lyases that act on specific adenines in single-stranded DNA.

Although the biological function of DNA glycosylases is to protect the genome by removal of potentially cytotoxic or mutagenic bases, this investigation describes the existence of natural DNA glycosylases with activity on undamaged, nonmispaired bases. Gelonin, pokeweed antiviral protein, and ricin, previously described as ribosome-inactivating proteins, are shown to damage single-stranded DNA by removal of a protein-specific set of adenines and cleavage at the resulting abasic sites. Using an oligonucleotide as the substrate reveals that the reaction proceeds via the enzyme-DNA imino intermediate characteristic of DNA glycosylase/AP lyases. The adenine glycosylase activity on single-stranded DNA reported here challenges the concept that a normal base has to be in a mismatch to be specifically removed. By contrast to other glycosylases, these enzymes are expected to damage DNA rather than participate in repair processes. The significance of this DNase activity to the biological function of these plant proteins and to their toxicity to animal cells remains to be determined.

Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin.

Several plant ribotoxins, including gelonin, were reported to have additional weak nuclease activities on supercoiled DNA. The potential contribution of this activity to their cytotoxicity has not been given serious consideration due to concerns about contaminating nucleases in the protein preparations. We now report the degradation of single-stranded DNA by preparations of native plant gelonin and recombinant gelonin produced in E. coli. The DNase activity of both preparations is similarly modulated by zinc. An SDS-PAGE DNase assay identifies gelonin as the polypeptide responsible for deoxyribonuclease activity.

Electrophysiology and morphology of neurons in rat perirhinal cortex.

The intrinsic membrane properties of perirhinal cortical neurons were studied by intracellular recording in in vitro rat brain slices. Gross morphology was also examined through injection of the fluorescent dye carboxyfluorescein. The cells encountered displayed a diversity of electrophysiological properties, and were similar to cells reported in other neocortical areas with regard to spiking patterns, afterpotentials, and morphology. However, very few (4%) intrinsically bursting neurons were encountered. Two pyramidal cells with thick apical dendrites were filled, and both fired doublets of action potentials for their first suprathreshold events. Of the filled pyramidal cells with thin apical dendrites, most (9/11) fired single action potentials for their first suprathreshold events. A variety of classification schemes were used to group the data, and several schemes were found to be equally successful. According to one of the schemes, cells recorded with carboxyfluorescein filled electrodes had significantly greater action potential widths at half-amplitude and more depolarized resting potentials than cells recorded without this dye.