Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb

In the olfactory bulb (OB) dopaminergic (DA) neurones constitute a fraction of the cells occupying the most external (glomerular) layer (Halász et al.1977). In this region, populated by three types of interneurons, periglomerular (PG) cells, short-axon cells and external tufted (ET) cells (Halász1990) - often collectively referred to as juxtaglomerular cells - an estimated 10% of the neurones in adulthood are positive for tyrosine hydroxylase (TH) (McLean and Shipley1988; Kratskin and Belluzzi2003), the rate limiting enzyme for dopamine synthesis. Dopaminergic neurones in the glomerular layer include PG cells (Gall et al.1987; Kosaka et al.1985) and a fraction of ET cells (Halász1990). Several studies have focused on the role of dopamine in the olfactory bulb, using immunohistochemical (Baker et al.1983; Guthrie et al.1991), behavioral (Doty and Risser1989), and electrophysiological techniques (Nowycky et al.1983; Ennis et al.2001; Davila et al.2003). The more complete description of the functional properties of DA neurons in the OB is probably the paper of Pignatelli (Pignatelli et al.2005), but it was incomplete, as it did not consider the contribution of the inward rectifier currents, a lacuna which is filled in the present work. A property shared by many DA neurons in the CNS is their capacity to generate rhythmic action potentials even in the absence of synaptic inputs (Grace and Onn1989; Hainsworth et al.1991; Yung et al.1991; Feigenspan et al.1998; Neuhoff et al.2002). In this paper we show for the first time that DA cells in the glomerular layer of the olfactory bulb possess a pacemaker activity, and we provide an explanation for the ionic basis of rhythm generation in these cells. There is an additional reason to study the functional properties of DA neurones in the OB other than their role in olfaction. The olfactory bulb is one of the rare regions of the mammalian CNS in which new cells, derived from stem cells in the anterior subventricular zone, are also added in adulthood (Gross2000). In the OB, these cells differentiate in interneurones in the granular and glomerular layers. Among these cells there are DA neurones (Betarbet et al.1996; Baker et al.2001), and this has raised a remarkable interest because, for their accessibility, they could provide a convenient source of autologous DA neurons for transplant therapies in neurodegenerative diseases, like Parkinson’s disease

Dopaminergic Neurones in the Main Olfactory Bulb: An Overview from an Electrophysiological Perspective

The olfactory bulb (OB), the first center processing olfactory information, is characterized by a vigorous life-long activity-dependent plasticity responsible for a variety of odor-evoked behavioral responses. It hosts the more numerous group of dopaminergic (DA) neurones in the central nervous system, cells strategically positioned at the entry of the bulbar circuitry, directly in contact with the olfactory nerve terminals, which play a key role in odor processing and in the adaptation of the bulbar network to external conditions. Here, we focus mainly on the electrophysiological properties of DA interneurones, reviewing findings concerning their excitability profiles in adulthood and in different phases of adult neurogenesis. We also discuss dynamic changes of the DA interneurones related to environmental stimuli and their possible functional implications

A five-conductance model of the action potential in the rat sympathetic neurone

none2The origin of the action potential in neurones has yet to be answered satisfactorily for most cells. We present here a five-conductance model of the somatic membrane of the mature and intact sympathetic neurone studied in situ in the isolated rat superior cervical ganglion under two-electrode voltage-clamp conditions. The neural membrane hosts five separate types of voltage-dependent ionic conductances, which have been isolated at 37 degrees C by using simple manipulations such as conditioning-test protocols and external ionic pharmacological treatments. The total current could be separated into two distinct inward components: (1) the sodium current, INa, and (2) the calcium current, ICa; and three outward components: (1) the delayed rectifier, IKV, (2) the transient IA, and (3) the calcium-dependent IKCa. Each current has been kinetically characterized in the framework of the Hodgkin-Huxley scheme used for the squid giant axon. Continuous mathematical functions are now available for the activation and inactivation (where present) gating mechanisms of each current which, together with the maximum conductance values measured in the experiments, allow for a satisfactory reconstruction of the individual current tracings over a wide range of membrane voltage. The results obtained are integrated in a full mathematical model which, by describing the electrical behaviour of the neurone under current-clamp conditions, leads to a quantitative understanding of the physiological firing pattern. While, as expected, the fast inward current carried by Na+ contributes to the depolarizing phase of the action potential, the spike falling phase is more complex than previous explanations. IKCa, with a minor contribution from IKV, repolarizes the neurone only under conditions of low cell internal negativity. Their role becomes less pronounced in the voltage range negative to -60 mV, where membrane repolarization allows IA to deinactivate. In the spike arising from these voltage levels the membrane repolarization is mainly sustained by IA, which proves to be the only current sufficiently fast and large enough to recharge the membrane capacitor at the speed observed during activity. Different modes of firing coexist in the same neurone and the switching from one to another is fast and governed by the membrane potential level, which makes the selection between the different voltage-dependent channel systems. The neurone thus seems to be prepared to operate within a wide voltage range; the results presented indicate the basic factors underlying the different discrete behaviours.noneO. BELLUZZI; O. SACCHIBelluzzi, Ottorino; Sacchi, Osca

Calretinin-periglomerular interneurons in mice olfactory bulb: Cells of few words

Within the olfactory bulb (OB), periglomerular (PG) cells consist of various types of interneurons, generally classified by their chemical properties such as neurotransmitter and calcium binding proteins. Calretinin (CR) characterizes morphologically and functionally the more numerous and one of the less known subpopulation of PG cells in the OB. Using of transgenic mice expressing eGFP under the CR promoter, we have tried to obtain the first functional characterization of these cells. Electrophysiological recordings were made in these cells using the patch-clamp technique in thin slices. Using ion substitution methods and specific blockers, we dissected the main voltage- dependent conductances present, obtaining a complete kinetic description for each of them. The more peculiar property of these cells from the electrophysiological point of view is the presence only of a single K-current, A-type - there is no trace of delayed rectifier or of Ca-dependent K-current. Other currents identified, isolated and fully characterized are a fast sodium current, a small L-type calcium current, and an inward rectifier, h-type cationic current. As a consequence of the peculiar complement of voltage-dependent conductances present in these cells, and in particular the absence of delayed-rectifier potassium currents, under the functional point of view these cells present two interesting properties. First, in response to prolonged depolarisations, after the inactivation of the A-current these cells behave as a purely ohmic elements, showing no outward rectification. Second, the CR cells studied can respond only with a single action potential to excitatory inputs; since they send inhibitory synapses to projection neurones, they seem to be designed to inhibit responses of the main neurones to isolated, random excitatory signals, rapidly losing their vetoing effect in response to more structured, repetitive excitatory signals. We propose that a possible role for these rather untalkative interneurons in the intense exchange of messages within the OB might be that of improving the signal-to-noise ratio in the first stages of the olfactory information processing

Sodium current in periglomerular cells of rat olfactory bulb in vitro.

none2The capacity of periglomerular cells (PGc) to give fast, Na-dependent action potentials is a crucial and debated issue for the comprehension of how sensory information is processed in the olfactory bulb (OB). Using patchclamp whole cell recording in thin slices of rat OB (P8-P20) we showed that fast sodium conductance is present in all the PGc studied, that this current is sufficiently large to generate action potentials and that action potentials can be evoked in these cells by direct stimulation of the olfactory nerve. A comprehensive kinetic characterization of INa is also presented.nonePuopolo M; Belluzzi O.Puopolo, M; Belluzzi, Ottorin

Inhibitory synapses among interneurons in the glomerular layer of rat and frog olfactory bulbs.

none2The patch-clamp technique was applied to periglomerular (PG) cells from slices of frog and rat olfactory bulbs to characterize whole cell and single-channel properties of inhibitory synaptic currents. Spontaneous and electrically driven bicuculline-sensitive synaptic events were recorded under ionic conditions that excluded any possible interference of excitatory synapses. The peak amplitude distribution of spontaneous events could be fitted by several Gaussians having the same interpeak distance. Spontaneous currents reversed polarity at the chloride equilibrium potential and were suppressed by 2 mM Co2+; the decay phase could be fitted with a single exponential having a time constant of approximately 10 ms at 0 mV. Bicuculline-sensitive monosynaptic responses could be evoked in PG cells by electrical stimulations delivered at the distance of several glomeruli within the glomerular layer. Finally, in excised outside-out patches, single-channel analysis revealed the presence of typical gamma-aminobutyric acid-A receptor channels, with a single-channel conductance of 28 pS in symmetrical chloride and mean open times of 3-4 ms. The simplest explanation of these data, effectively supported by pristine anatomic findings, is that there could be inhibitory contacts among interneurons in the glomerular layer.nonePuopolo M; Belluzzi O.Puopolo, M; Belluzzi, Ottorin

Modifications of A-current kinetics in mammalian central neurones induced by extracellular zinc.

none21. Whole-cell voltage clamp recordings were used to study the action of the transition ion zinc on the A-current kinetics in granule cells from rat cerebellar slices. 2. The effects of zinc have been tested in the concentration range from 1 microM to 1 mM, and fully characterized on all kinetic parameters at 100 and 300 microM. All the effects observed were rapid, concentration dependent and fully reversible. 3. Steady-state inactivation curves are strongly shifted towards depolarized potentials, with activation curves much less so. These shifts lead to an increase of the peak current amplitude around physiological resting membrane potentials and to a decrease at hyperpolarized potentials. 4. The forward 'on' rate constants are slowed by Zn2+ at a concentration of 100-300 microM by a factor from 1.5 to 4. The backward 'off' rate constants are unaffected by Zn2+. 5. The development of IA inactivation, as measured from the current decay, is not affected by Zn2+ up to 1 mM. Removal of inactivation is, on the contrary, significantly slowed. 6. The results are neither compatible with the theory of the surface charge screening effect nor with a mechanism involving channel block. It seems more likely that Zn2+ interferes with the channel gating by binding to a specific domain of the channel protein. 7. After treatment with Hg2+, which is irreversible, Zn2+ still maintains its effects, which suggest that the two divalents act at different sites. 8. In view of the widespread distribution of zinc throughout the brain, its actions on the A-current could play an important role in physiological function.noneBardoni R.; Belluzzi O.Bardoni, R.; Belluzzi, Ottorin

Hyperpolarisation-activated current in glomerular cells of the rat olfactory bulb

Whole-cell patch-clamp recordings were carried out in visually identified periglomerular and external tufted cells of rat olfactory bulb. Most of the neurones showed a slowly developing hyperpolarisation-activated current with a threshold generally positive to resting potential and with a strongly voltage-dependent activation time constant. The current, identified as Ih, was sodium- and potassium-sensitive, suppressed by external caesium, and insensitive to barium. Under current-clamp conditions, perfusion with caesium induced a 10 mV hyperpolarisation and a marked reduction of the rate of low-frequency oscillations induced experimentally. It is concluded that most of the cells in the rat glomerular layer present a distinct h-current, which is tonically active at rest and which may contribute to the oscillatory behaviour of the bulbar network

Functional heterogeneity of periglomerular cells in the rat olfactory bulb.

none2The periglomerular cells of the rat olfactory bulb, a virtually unknown population of interneurons, have been studied applying the whole-cell patch-clamp technique to thin slices. A prominent result, obtained under current-clamp conditions, is that these cells appear to be functionally heterogeneous, and show distinct excitability profiles. Voltage-clamp analysis allows the identification of the ionic basis of these differences and suggests a division into at least two classes, based on the characteristics of the K+ conductances. The first group displays two K+ conductances (delayed rectifier, gKV, and fast transient, gA) of similar amplitude, and under current-clamp conditions shows the usual outward rectifying behaviour at depolarized potentials. The second group has a large gA, and a small or absent gKV. Consequently, following sustained depolarizations under current-clamp conditions leading to inactivation of gA, these neurons do not show any sign of outward rectification and behave as ohmic elements, as normally observed only at hyperpolarized potentials. The transition ion zinc (10-300 microM) affects gA but not gKV The inactivation process (steady-state curve and rate constant) is strongly altered by Zn2+, the activation process less so; open-channel conductance is not affected. The Zn2+ effect is unlikely to be due to surface charge screening or to a mechanism involving channel block. In view of the substantial presence of zinc ions in the olfactory nerve terminals, its actions on the A-current could be of some relevance for physiological function.nonePuopolo M; Belluzzi O.Puopolo, M; Belluzzi, Ottorin

Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices.

none21. Whole-cell voltage-clamp techniques were used to study voltage-activated transient potassium currents in a large sample (n = 143) of granule cells (GrC) from rat cerebellar slices. Tetrodotoxin (TTX; 0.1 microM) was used to block sodium currents, while calcium current was too small to be seen under ordinary conditions. 2. Depolarizing pulses from -50 mV evoked a slow, sustained outward current, developing with a time constant of 10 ms, inactivating over a time scale of seconds and which could be suppressed by 20 mM tetraethylammonium (TEA). By preventing the Ca2+ inflow, this slow outward current could be further separated into a Ca(2+)-dependent and a Ca(2+)-independent component. 3. After conditioning hyperpolarizations to potentials negative to -60 mV, depolarizations elicited transient outward current, peaking in 1-2 ms and inactivating rapidly (approximately 10 ms at 20 degrees C), showing the overall kinetic characteristics of the A-current (IA). The current activated following third-order kinetics and showed a maximal conductance of 12 nS per cell, corresponding to a normalized conductance of 3.8 nS/pF. 4. IA was insensitive to TEA and to the Ca(2+)-channel blockers. 4-Aminopyridine (4-AP) reduced the A-current amplitude by approximately 20%, and the delayed outward currents by > 80%. 5. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzmann function with a slope factor of 8.4 mV and half-inactivation occurring at -78.8 mV. Activation of IA was characterized by a Boltzmann curve with the midpoint at -46.7 mV and with a slope factor of 19.8 mV. 6. IA activation and inactivation was best fitted by the Hodgkin-Huxley m3h formalism. The rate of activation, tau a, was voltage-dependent, and had values ranging from 0.55 ms at -40 mV to 0.2 ms at +50 mV. Double-pulse experiment showed that development and removal of inactivation followed a single-exponential time course; the inactivation time constant, tau ha, was markedly voltage-dependent and ranged from approximately 10 ms at -40 and -100 mV and 70 ms at -70 mV. 7. A set of continuous equations has been developed describing the voltage-dependence of both the steady-state and time constant of activation and inactivation processes, allowing a satisfactory numerical reconstruction of the A-current over the physiologically significant membrane voltage range.(ABSTRACT TRUNCATED AT 400 WORDS)noneBardoni R.; Belluzzi O.Bardoni, R.; Belluzzi, Ottorin