Developmental changes in the expression of low-voltage-activated Ca2+ channels in rat visual cortical neurones

The functional properties of low-voltage-activated (LVA) Ca2+ channels were studied in pyramidal neurones from different rat visual cortical layers in order to investigate changes in their properties during early postnatal development. Ca2+ currents were recorded in brain slices using the whole-cell patch-clamp technique in rats from three age groups: 2, 3 and 12 days old (postnatal day (P) 2, P3 and P12). It was demonstrated that LVA Ca2+ currents are present in neurones from superficial (I-II) and deep (V-VI) visual cortex layers of P2 and P3 rats. No LVA Ca2+ currents were observed in neurones from the middle (III-IV) layers of these rats. The LVA Ca2+ currents observed in P2 and P3 neurones from both superficial and deep layers could be completely blocked by nifedipine (100 μM) and were insensitive to Ni2+ (25 μM). The density of LVA Ca2+ currents decreased rapidly during the early stages of postnatal development, while the density of high-voltage-activated (HVA) Ca2+ currents progressively increased up to the twelfth postnatal day. No LVA Ca2+ currents were found in P12 neurones from any of the layers. Only HVA Ca2+ currents with high sensitivity to F− applied through the patch pipette were observed. The kinetics of LVA Ca2+ currents could be well approximated by the m2h Hodgkin-Huxley equation with an inactivation time constant of 24 ± 6 ms. The steady-state inactivation curve fitted by a Boltzmann function had the following parameters: membrane potential at half-inactivation, -86.9 mV; steepness coefficient,3.4 mV. It is concluded that, in visual cortical neurones, LVA Ca2+ channels are expressed only in the neurones of deep and superficial layers over a short period during the earliest postnatal stages. These channels are nifedipine sensitive and similar in functional properties to those in the laterodorsal (LD) thalamic nucleus. However, the cortical neurones do not express another (‘slow’) type of LVA Ca2+ channel, which is permanently present in LD thalamic neurones after the second postnatal week, indicating that the developmental time course of cortical and thalamic cellsdifferent. The functional properties of nerve cells in different parts of the nervous system are determined by the specificity of their morphological structure as well as by the composition of their voltage- and receptor-operated ion channels. The expression of channel proteins is extremely variable in different neurones and highly dependent on the developmental stage of the animal (Thompson & Wong, 1991; Fedulova, Kostyuk & Veselovsky, 1994). Some of these proteins appear only for a short period, being responsible for triggering the differentiation of neuronal precursors and the establishment and maturation of neuronal connections, while others form a constant basis for the activity of neuronal networks. Analysis of the expression and functioning of voltage-operated Ca2+ channels (VOCCs) is especially important for the understanding of these processes, as Ca2+ ions are known to play a critical role in the formation of the nervous system (Al-Mohanna, Cave & Bolsover, 1992; Amato, Al-Mohanna & Bolsover, 1996), and to be the main messengers in synaptic transmission at all neuronal junctions. Of especial interest in this respect is the presence of low-voltage-activated (LVA) Ca2+ channels, which can be activated at voltages close to the membrane resting potential provided that their steady-state inactivation is removed by some preceding hyperpolarizing influences, and which can therefore easily generate the spontaneous Ca2+ transients necessary for morphogenesis. At present, data on the development of expression of such channels in different neurones are extremely variable. Thus, during artificially induced differentiation in neuronal tumour cell lines, only LVA Ca2+ channels appear at the start of differentiation, and then decrease as other types of ion channels are expressed (Veselovsky & Fomina, 1986). In rat and mouse dorsal root ganglion neurones LVA (T-type) Ca2+ channels show a peak of expression just around the time of birth, while later on their density starts to decline until their complete disappearance in most neurones beyond the first few weeks (Fedulova, Kostyuk & Veselovsky, 1986; Fedulova et al. 1994). In cultured Xenopus sensory neurones they are expressed only during the first 20-40 h of culture (Barish, 1991). The same is true for neurones cultured from adult or embryonic rat and guinea-pig hippocampal pyramidal neurones (O'Dell & Alger, 1991; Thompson & Wong, 1991) and rat neostriatum (Bargas, Surmeier & Kitai, 1991). In contrast to this, however, LVA Ca2+ channels are the predominant type of VOCC in certain thalamic and hypothalamic neurones and are a constant feature of the excitability mechanism, playing an important role in the organization of slow rhythmic activity (Akaike, Kostyuk & Osipchuk, 1989; Huguenard & Prince, 1992). The developmental appearance of such channels is quite complicated. Thus, the population of LVA Ca2+ channels in neurones of the rat laterodorsal (LD) thalamic nucleus was found to be homogeneous in kinetic and pharmacological properties only during the first postnatal week. The corresponding Ca2+ currents demonstrated fast inactivation kinetics with a monoexponential time course (∼30 ms) and high sensitivity to nifedipine (Kd= 2.6 μM). However, from the second postnatal week onwards, a more slowly inactivating component appeared in LVA Ca2+ currents from the same neurones, which showed insensitivity to nifedipine but high sensitivity to Ni2+ (Tarasenko, Kostyuk, Eremin & Isaev, 1997). The two types of LVA Ca2+ channel are probably associated with different neuronal functions, as the appearance of the ‘slow’ channels (with an inactivation time constant of ∼60 ms) coincided with development of the dendritic tree in the corresponding cells. These findings stimulated us to analyse in more detail the kinetic, pharmacological and developmental properties of LVA Ca2+ channels in neocortical neurones closely connected functionally to the neuronal activity of associative thalamic nuclei. The presence of LVA Ca2+ currents has been reported in pyramidal neurones in slices from the rat visual cortex (Franz, Galvan & Constanti, 1986; Sutor & Zieglgansberger, 1987) and in pyramidal neurones acutely isolated from the rat sensorimotor cortex (Sayer, Schwindt & Crill, 1990). However, the properties of these channels have not as yet been fully characterized. The cellular heterogeneity of the cortex and the difficulty of identifying specific cell types has substantially limited the study of characteristics and developmental changes. This complexity is evident from a recent study of pyramidal neurones from guinea-pig medial frontal cortex, which found that only cells located lower than 500 μm from the pial surface (approximately layers V-VI) were able to generate LVA Ca2+ currents and low-threshold Ca2+ spikes (LTS) (De la Pena & Geijo-Barrientos, 1996). In the present study we attempted to investigate this problem further by using neurones from different layers of the rat visual cortex during the first 12 days of postnatal development.