Electrophysiological and morphological heterogeneity of neurons in slices of rat suprachiasmatic nucleus

Abstract

1. Whole cell patch clamp recordings of neurons in slices of the suprachiasmatic nucleus (SCN) were made in order to assess their electrophysiological and morphological heterogeneity. This assessment was accomplished by (i) quantification of intrinsic membrane properties recorded in current clamp mode, (ii) studying frequency distributions of these properties, (iii) grouping of cells based on visual inspection of data records, and (iv) use of cluster analysis methods. 2. Marked heterogeneity was found in the resting membrane potential, input resistance, time constant, rate of frequency adaptation, size of rebound depolarization (low-threshold Ca2+ potential) and regularity of firing. The frequency distribution of these membrane properties deviated significantly from a normal distribution. Other parameters, including spike amplitude and width, amplitude and rising slope of the spike after-hyperpolarization (AHP) and amplitude of the spike train AHP, showed considerable variability as well but generally obeyed a normal distribution. 3. Visual inspection of the data led to partitioning of cells into three clusters, viz. cluster I characterized by monophasic spike AHPs and irregular firing in the frequency range from 1.5 to 5.0 Hz; cluster II with biphasic spike AHPs and regular firing in the same range; and cluster III with large rebound depolarizations and biphasic spike AHPs. In a post hoc analysis, these clusters also appeared to differ in other membrane properties. This grouping was confirmed by hierarchical tree clustering and multidimensional scaling. 4. The light microscopic properties of recorded neurons were studied by biocytin labelling. Neurons had monopolar, bipolar or multipolar branching patterns and were often varicose. Axons sometimes originated from distal dendritic segments and usually branched into multiple collaterals. Many cells with extra-SCN projections also possessed intranuclear axon collaterals. We found no morphological differences between clusters except that cluster III neurons possessed more axon collaterals than cluster I or II cells. 5. These results suggest that SCN neurons are heterogeneous in some basic as well as active membrane properties and can be partitioned into at least three clusters. Cluster I and II cells fire spontaneously in a regular and irregular mode, respectively, and sustain prolonged spike trains. In contrast, cluster III cells have low firing rates but may adopt a burst-like firing mode when receiving appropriate input. While all clusters transmit output to target cells within and outside SCN, cluster III cells in particular are suggested to affect excitability of large numbers of SCN neurons by their extensive local network of axon collaterals.

Effects of Sodium Pump Activity on Spontaneous Firing in Neurons of the Rat Suprachiasmatic Nucleus

“…On average, zero K ϩ increased the rate of depolarization from 0.28 Ϯ 0.04 mV/ms (n ϭ 7) to 0.57 Ϯ 0.04 mV/ms (n ϭ 7), enhanced the peak AHP by 6.1 Ϯ 1.0 mV (n ϭ 7), but negligibly lowered the threshold potential by 0.1 Ϯ 0.3 mV (n ϭ 7). The zero K ϩ -induced increase in the rate of depolarization is consistent with the induction of an inward current caused by the blockade of sodium pump, whereas the increase of peak AHP suggests that zero K ϩ additionally potentiates the K ϩ currents responsible for generating the AHP in SCN neurons (Cloues and Sather 2003;Kim and Dudek 1993;Pennartz et al 1998;Teshima et al 2003;Thomson and West 1990;Wheal and Thomson 1984). The larger peak AHP in zero K ϩ might have prevented the partial inactivation of sodium channels that otherwise raised the firing thresholds and reduced the spike amplitudes as seen in the presence of 30 M strophanthidin (Fig.…”

“…Although the nature of the current that is potentiated by zero K ϩ is not known, the calcium-dependent potassium current is likely involved. In the cluster I neurons of the SCN (Pennartz et al 1998), at least three subtypes of Ca 2ϩ -activated K ϩ channels-apamin-sensitive channels, iberiotoxin-sensitive channels, and channels insensitive to both antagonistscontribute to the monophasic AHP (Cloues and Sather 2003). Similarly, the apamine-sensitive small-conductance Ca 2ϩ -activated K ϩ channel contributes to the "train" afterhyperpolarization after a train of action potentials in response to a depolarizing current (Teshima et al 2003).…”

Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis

“…These methods have recently been used to more objectively characterize neuronal populations in several brain areas such as neocortex and suprachiasmatic nucleus (Azouz et al, 1997, Pennartz et al, 1998, Cauli et al, 2000, Halabisky et al, 2004. A key element of cluster analysis is the selection of variables (electrophysiological parameters) to be considered in creating different subgroups.…”

“…The firing behavior of the neuron was first inspected visually for heterogeneity and later analyzed via unsupervised clustering analysis. This technique can assist in uncovering data structure and providing a scheme for classifying neuronal subtypes (Pennartz et al, 1998, Nowak et al, 2003, Halabisky et al, 2004. This method consists of plotting individual objects (neurons) in multidimensional space according to similarities or dissimilarities of input Pennartz et al, 1998, Cauli et al, 2000, Nowak et al, 2003.…”

“…The neurons spatially closest to each other are grouped together as a cluster. The analysis was performed using Ward's method which is based on the sum of squares between the clusters (Euclidean distances, summed over all variables) (Anderson et al, 1998) allowing an objective segregation of cell groups as previously reported (Pennartz et al, 1998, Cauli et al, 2000, Nowak et al, 2003. Electrophysiological variables were normalized to a common range (0-1) before performing the multidimentional analysis.…”