Determination of protein concentration in raw milk by mid-infrared fourier transform infrared/attenuated total reflectance spectroscopy.

This study investigates the potential use of attenuated total reflectance spectroscopy in the mid-infrared range for determining protein concentration in raw cow milk. The determination of protein concentration is based on the characteristic absorbance of milk proteins, which includes 2 absorbance bands in the 1500 to 1700 cm(-1) range, known as the amide I and amide II bands, and absorbance in the 1060 to 1100 cm(-1) range, which is associated with phosphate groups covalently bound to casein proteins. To minimize the influence of the strong water band (centered around 1640 cm(-1)) that overlaps with the amide I and amide II bands, an optimized automatic procedure for accurate water subtraction was applied. Following water subtraction, the spectra were analyzed by 3 methods, namely simple band integration, partial least squares (PLS) and neural networks. For the neural network models, the spectra were first decomposed by principal component analysis (PCA), and the neural network inputs were the spectra principal components scores. In addition, the concentrations of 2 constituents expected to interact with the protein (i.e., fat and lactose) were also used as inputs. These approaches were tested with 235 spectra of standardized raw milk samples, corresponding to 26 protein concentrations in the 2.47 to 3.90% (weight per volume) range. The simple integration method led to very poor results, whereas PLS resulted in prediction errors of about 0.22% protein. The neural network approach led to prediction errors of 0.20% protein when based on PCA scores only, and 0.08% protein when lactose and fat concentrations were also included in the model. These results indicate the potential usefulness of Fourier transform infrared/attenuated total reflectance spectroscopy for rapid, possibly online, determination of protein concentration in raw milk.

Highly 4-aminopyridine sensitive delayed rectifier current modulates the excitability of guinea pig cerebellar Purkinje cells.

The effects of low concentrations of 4-aminopyridine (4-AP) on the membrane properties of guinea pig cerebellar Purkinje cells were investigated in slice preparation using intracellular recordings. It was found that 1-10 microM 4-AP did not affect the resting potential or the input resistance of the cells, but reduced markedly the duration of the slowly depolarizing potential (SDP), and thus the latency to the firing of Ca2+ spikes in response to intracellular current pulses. Intradendritic recordings in the presence of tetrodotoxin, Cd2+, and low [Ca2+]o, which blocked all the regenerative responses, exhibited prominent membrane outward rectification in response to depolarizing current pulses. Under these conditions, the SDP was abolished and, in contrast, a slowly developing hyperpolarization was consistently observed. Application of 10 microM 4-AP reduced the outward membrane rectification in a reversible manner, but did not affect the transient hyperpolarization, which is usually attributed to the activation of potassium "A" current. These results demonstrate, for the first time, the presence of a highly 4-AP sensitive delayed rectifier in guinea pig cerebellar Purkinje cells, which prominently affects their excitability. The results also indicate that the slowly depolarizing potential of guinea pig Purkinje cells does not involve inactivation of transient potassium currents, which has been suggested previously as an underlying mechanism for this phenomenon in turtle Purkinje cells.

Period doubling of calcium spike firing in a model of a Purkinje cell dendrite.

Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained current injection, the cell firing pattern can move from tonic firing of Ca(2+) spikes to doublet firing and even to quadruplet firing or more complex firing. These firing patterns are not modified substantially if Na(+) currents are blocked. We show that the experimental results can be viewed as a slow transition of the neuronal dynamics through a period-doubling bifurcation. To further support this conclusion and to understand the underlying mechanism that leads to doublet firing, we develop and study a simple, one-compartment model of Purkinje cell dendrite. The neuron can also exhibit quadruplet and chaotic firing patterns that are similar to the firing patterns that some of the Purkinje cells exhibit experimentally. The effects of parameters such as temperature, applied current, and potassium reversal potential in the model resemble their effects in experiments. The model dynamics involve three time scales. Ca(2+)- dependent K(+) currents, with intermediate time scales, are responsible for the appearance of doublet firing, whereas a very slow hyperpolarizing current transfers the neuron from tonic to doublet firing. We use the fast-slow analysis to separate the effects of the three time scales. Fast-slow analysis of the neuronal dynamics, with the activation variable of the very slow, hyperpolarizing current considered as a parameter, reveals that the transitions occurs via a cascade of period-doubling bifurcations of the fast and intermediate subsystem as this slow variable increases. We carry out another analysis, with the Ca(2+) concentration considered as a parameter, to investigate the conditions for the generation of doublet firing in systems with one effective variable with intermediate time scale, in which the rest state of the fast subsystem is terminated by a saddle-node bifurcation. We find that the scenario of period doubling in these systems can occur only if (1) the time scale of the intermediate variable (here, the decay rate of the calcium concentration) is slow enough in comparison with the interspike interval of the tonic firing at the transition but is not too slow and (2) there is a biostability of the fast subsystem of the spike-generating variables.

Pressure-induced depression of synaptic transmission in the cerebellar parallel fibre synapse involves suppression of presynaptic N-type Ca2+ channels.

High pressure induces CNS hyperexcitability while markedly depressing synaptic transmitter release. We studied the effect of pressure (up to 10.1 MPa) on the parallel fibre (PF) synaptic response in biplanar cerebellar slices of adult guinea pigs. Pressure mildly reduced the PF volley amplitude and to a greater extent depressed the excitatory field postsynaptic potential (fPSP). The depression of the PF volley was noted even at supramaximal stimulus intensities, indicating an effect of pressure on the amplitude of the action potential in each axon. Low concentrations of TTX mimicked the effects of pressure on the PF volley without affecting the fPSP. Application omega-conotoxin GVIA (omega-CgTx) reduced the synaptic efficacy by 34.3+/-2.7%. However, in the presence of omega-CgTx the synaptic depression at pressure was significantly reduced. Reduced Ca2+ entry by application of Cd2+ or low [Ca2+]o did not have a similar influence on the effects of pressure. Application of omega-AGA IVA, omega-AGA TK and Funnel-web spider toxin did not affect the synaptic response in concentrations that usually block P-type Ca2+ channels, whilst the N/P/Q-type blocker omega-conotoxin MVIIC reduced the response to 52.7+/-5.0% indicating the involvement of Q-type channels and R-type channels in the non-N-type fraction of Ca2+ entry. The results demonstrate that N-type Ca2+ channels play a crucial role in the induction of PF synaptic depression at pressure. This finding suggests a coherent mechanism for the induction of CNS hyperexcitability at pressure.

Spontaneous Na+ and Ca2+ spike firing of cerebellar Purkinje neurons at high pressure.

The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guinea-pig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na+ spike parameters or the frequency of regular Na+ spike firing. When Na+ currents were blocked by 0.5-1 microM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca2+ spike amplitude to 90.2+/-3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca2+ spike were even less prominent when K+ currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca2+ spike firing to 152.2+/-10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9+/-8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K+]o) and 1 microM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca2+]o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca2+]o slightly increased the firing of doublets. These results show that the single somatic Na+ spike is insensitive and the dendritic Ca2+ spike is only mildly sensitive to pressure. However, alterations in Ca2+ spike firing pattern suggest that modulation of dendritic K+ currents induce depression of dendritic excitability at pressure.

Potassium currents modulation of calcium spike firing in dendrites of cerebellar Purkinje cells.

The pattern of sustained Ca2+ spike firing was investigated, using macropatch clamp and intracellular recordings, in guinea pig cerebellar Purkinje cells. Under our standard experimental conditions (30 degrees C, 5 mM [K+]o, 2 mM [Ca2+]o, 1 microM tetrodotoxin), each firing period started with uniform firing and gradually turned into a doublet pattern with a large spike afterhyperpolarization (AHP) between the doublets. Macropatch clamp recordings from localized dendritic regions revealed that each doublet is composed of two similar inward current deflections. This result indicated, for both peaks, an active process in the recording site and contradicted the possibility that they reflect firing in two completely separated dendritic regions. When [K+]o was increased the transition to a doublet pattern occurred earlier and the doublets became more pronounced. A similar but more prominent effect occurred following application of 1-10 microM 4-aminopyridine, which also reduced the threshold, increased the spike amplitude, and shortened the initial delay of evoked Ca2+ spike firing. In contrast, membrane depolarization, increased [Ca2+]o, and application of quinidine (but not apamine) markedly suppressed the generation of doublet pattern. During uniform initial firing, a short hyperpolarizing pulse that mimicked a large AHP induced a subsequent doublet. A short depolarizing pulse following a single spike induced an artificial doublet followed by a large AHP. These results indicate that the pattern of Ca2+ spike firing in the dendrites of Purkinje cells is dynamically modulated by a highly aminopyridine-sensitive K+ current, and probably also by a Ca2+-activated potassium current.