- Email: [email protected]
Network Experimental Approaches
C H A P T E R
5 Network Experimental Approaches: Ex vivo Recording Victor V. Uteshev Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA
INTRODUCTION
DIRECT SYNCHRONIZATION VIA CHEMICAL AND ELECTRICAL SYNAPSES IN THE OLFACTORY BULB
In a functional brain, contributions of individual neurons can often be most effective if the neuronal activity is synchronized. Synchronization can be achieved either by a direct communication among neurons or by inhibition of asynchrony (i.e. reducing the chances of individual neurons to act differently from the rest of the group). For example, in the olfactory bulb, a direct communication among neurons via synaptic connections and gap junctions1 prevents external tufted (ET) cells of the same glomerulus from behaving asynchronously with the rest of the neuronal population, while natural pacemaker neurons of the hypothalamic histaminergic tuberomammillary (TM) nucleus that are electrically independent from one another (as discussed in this chapter) can be synchronously shut off during sleep by GABAergic inputs from the ventrolateral preoptic nucleus.2 Therefore, in some cases, the impact of individual neurons on the network status can be deduced either from understanding of the network structure as well as synaptic and/or gap junction connections, or from knowledge of pharmacology and kinetics of the external inputs and neuronal receptors involved in signaling. Neurons, on the other hand, may define the level of sustained excitability of neuronal networks, network stability, and sensitivity to the action of endogenous and exogenous compounds. Although “in vitro” is a term that has been often applied to brain slice work, it is perhaps more accurate to label acute slice work as “ex vivo”, which is done in this chapter. The term “in vitro” is used to refer to cultured neurons and cell lines.
Neuronal Networks in Brain Function, CNS Disorders, and Therapeutics http://dx.doi.org/10.1016/B978-0-12-415804-7.00005-8
The olfactory bulb is a complex and intriguing brain structure involved in encoding, transfer, processing, and decoding of odorant-evoked sensory information. It has a multifaceted network of interneuronal connections that include dendrodendritic chemical synapses and electrical synapses, also known as gap junctions.3 Organization and properties of neuronal and synaptic connections facilitate neuronal synchronization, which may be critical for effective odor discrimination.4e6 Moreover, optimal neuronal synchronization may enhance transmission of information from sensory receptors to higher order brain structures.7,8 Integration and processing of sensory olfactory information at the level of the olfactory bulb involve stimulation of a complex neuronal circuitry, specific patterns of activation of chemical and electrical synapses, and coordination of activity of multiple glomeruli. The glomeruli of the olfactory bulb serve as the sites of the first synapse for olfactory sensory inputs, which are processed by three types of glomerular juxtaglomerular (JG) neurons: ET, periglomerular (PG), and short axon (SA) cells. Olfactory bulb output neurons exhibit highly synchronized activity in acute ex vivo preparation of brain slices despite deprivation of their normal sensory inputs. Synchronous activity is common among output neurons whose tufted dendrites belong to the same glomerulus. Such synchronization was found among mitral cells,9 ET cells,1 tufted cells,10 and mitral cells.11 Furthermore, additional interactions among neurons within the same glomerulus or different glomeruli arise from
67 Copyright Ó 2014 Elsevier Inc. All rights reserved.
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FIGURE 5.1 Synchronous slow and fast Excitatory Post-Synaptic Currents (EPSCs) in ET cells of the same glomerulus. All data presented in this figure were obtained from the same ET cell pair. (A) Simultaneous extracellular recordings from two ET cells with correlated spike bursting (correlated spikes shown in the inset at right). (B) The same two cells were then recorded in whole-cell configuration in voltage clamp (HP of 60 mV) using electrodes (containing CsMeSO3 and QX-314). Note the synchronous slow (horizontal lines with arrows on both sides) and fast (asterisks; example shown at right) EPSCs. (C) Cross-correlogram of the spike trains (5 min recording sample, 1 ms bins) shows a significant correlation with a peak near zero lag time (see inset). (D) The cross-correlogram of the membrane current (50 s of intracellular recording sample, 2 ms bins) shows both a broad (small lines with arrows on both sides) and narrow (asterisk; see inset) correlation that peaked at zero time lag. The gray trace is the cross-correlogram of the membrane current traces after shifting the second trace by 5 s (to determine significance, see “Materials and Methods” in Ref. 1). (E) Cross-correlogram of the EPSC trains (5 min recording sample, 1 ms bins) shows a significant peak only in the bin at zero time lag, indicating synchronous EPSCs in the two recorded cells. The vertical axes in (CeE) demonstrate a cross-correlation value, C. (F) Cumulative probability histograms of the EPSC amplitudes of synchronous and asynchronous EPSCs recorded in both cells (during 5 min). The synchronous EPSCs exhibited significantly larger amplitude than asynchronous EPSCs (p < 0.0001, KeS test). (G) Scatterplot of the amplitude of synchronous EPSCs in cell 2 versus cell 1 and a linear regression fit showing a significant positive correlation. (H) Photograph of the two recorded cells after biocytin staining showing the overlap of dendrites in the same glomerulus (dashed line). Source: From Hayar et al., 2005,1 with permission from the Society for Neuroscience.
extensive lateral dendrites found in output neurons that establish dendrodendritic connections with interneurons.12 For example, a highly correlated activity was found among ET cells, SA cells, and PG cells13,14 and among mitral cells and granule cells.15 Using an ex vivo olfactory bulb slice preparation and dual patch-clamp recordings from the JG circuitry (Figure 5.1), it has been determined that ET cells spontaneously fire rhythmic spike bursts in the theta frequency
range and receive monosynaptic olfactory nerve input.1,16 By contrast, SA and most PG cells do not receive monosynaptic olfactory input, but receive instead monosynaptic excitatory inputs from ET cells. These observations demonstrated that ET cells serve as a major excitatory link between olfactory nerve input and other JG cells. Intriguingly, within the same glomerulus, spontaneous bursts among ET cells are highly correlated even in the absence of activity of fast chemical
INHIBITION OF ASYNCHRONY IN SPONTANEOUS ACTIVITY OF HYPOTHALAMIC HISTAMINERGIC TM NEURONS
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FIGURE 5.2 Simultaneous recordings from pairs of ventral TM (VTM) neurons in acute hypothalamic brain slices. Neighboring TM neurons (arrows) located within 200 mm from one another (A) were selected for simultaneous recordings of spontaneous action potentials (APs) in current-clamp patch-clamp experiments (BeC). The results obtained from analyzing nine pairs of TM neurons demonstrated random uncorrelated patterns of spontaneous APs of neighboring TM neurons. In each pair, spontaneous APs fired in one TM neuron were not detectable in the other TM neuron inhibited by injection of small (10 pA) hyperpolarizing currents (C).
synapses (i.e. when activity of GABAergic and glutamatergic fast synapses is blocked).1 Analysis of experimental data indicated that the synchronized spontaneous bursting of ET cells within the same glomerulus is coordinated by synaptic transmission and gap junction coupling among ET cells (Figure 5.1). Therefore, the synchronous bursting of ET cells and, thus, synchronized odor-evoked glomerular output may function to amplify transient sensory inputs and coordinate glomerular outputs to ensure that the high fidelity of the specific odorant profile information is relayed to higher order brain structures. Moreover, the bursting activity of ET cells is readily entrained by patterns of odor-evoked sensory inputs at sniffing frequencies.13 These results suggest that amplification of the sensory input by ET cells may help enhance synchronization of the glomerular network tuned to the sniffing of the animal and, thus, enhance its functionality. The presence of highly multifaceted neuronal interactions in the olfactory bulb circuitry may reflect a need of olfactory bulb neurons to tune their activity to the animal’s respiratory cycle17e19.
INHIBITION OF ASYNCHRONY IN SPONTANEOUS ACTIVITY OF HYPOTHALAMIC HISTAMINERGIC TM NEURONS The TM nucleus of the posterior hypothalamus represents the sole source of brain histamine and contributes to regulation of normal cognition, sleepewakefulness cycles, food and water consumption, and energy
metabolism (see Chapter 21).20,21 Histamine, a “waking substance”, regulates sleep and wakefulness, while bloodebrain barrier permeable histamine H1 receptor antagonists cause sedation. TM neurons are typical, natural pacemakers and demonstrate spontaneous firing in the absence of synaptic inputs in vivo,22,23 in vitro,24e26 and after acute dissociation.27,28 The firing pattern of TM neurons as well as the histamine concentration in cerebrospinal fluid follow a circadian rhythm (see Chapter 14) and depend on the state of arousal.29,30 The highest frequency of TM neuronal spontaneous firing (6e8 Hz) corresponds to wakefulness in vivo. The activity of TM neurons decreases during nonerapid eye movement (non-REM) sleep and ceases completely during rapid eye movement (REM) sleep.22,23 Blocking spontaneous firing by hypothalamic perfusion with tetrodotoxin inhibits histamine release,31,32 which has been shown to require extracellular Ca2þ in vivo33 and in vitro.31,34 The presence of both inactivating and noninactivating voltage-gated calcium channels in TM neurons35 produces a dynamic equilibrium supportive of sustained spontaneous firing via associated Ca2þ signaling.36,37 In contrast to olfactory ET cells, TM neurons do not communicate with their TM neighbors via chemical or electrical synapses (Figure 5.2). Instead, TM neurons fire action potentials independently and within a relatively narrow range (1e6 Hz) of frequencies unless inhibited, for instance, via GABAergic inputs from the ventrolateral preoptic nucleus.2 A strong relationship between the frequency of spontaneous firing and the corresponding Ca2þ influx in TM neurons has been established in ex vivo experiments
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FIGURE 5.3 Correlation among neuronal properties and function. (AeB) A typical example of simultaneous current-clamp (A) and Ca2þ (B) recordings from the same TM neuron. Spontaneous firing of TM neurons (A) correlates with elevation in [Ca2þ]i (B) measured using Ca2þ Green-1 dye included into the intracellular pipette solution. A graphic relationship between the frequency of spontaneous firing and Ca2þ influx (in mM and as a ratio of F/F0) is shown in (C). Firing frequencies (1e4 Hz) that are observed in vivo during wakefulness correspond to [Ca2þ]i w 0.2e1 mM and may support histamine release. (DeE) Caffeine inhibits spontaneous firing of TM neurons (D) by stimulating Ca2þ release from Ca2þ stores (E) and activation of Ca2þ-dependent potassium conductance (KCa). Both an outward conductance measured in
STOCHASTIC ENHANCEMENT OF NEURONAL EXCITABILITY: CAN A SINGLE ION CHANNEL EXCITE THE ENTIRE NEURON?
utilizing simultaneous Ca2þ imaging and electrophysiological patch-clamp recordings from TM neurons in acute brains slices.25 As the firing pattern of TM neurons defines the rate of histamine release and arousal state in mammals,22,23,31,32 this correlation between spontaneous firing and [Ca2þ]i predicts the levels of cytosolic Ca2þ that correspond to specific behavioral states (e.g. sleep or wakefulness) (Figure 5.3(A)e(C)). In particular, the maximum firing frequency observed in these ex vivo experiments was w4 Hz.25 This frequency corresponds to wakefulness in vivo22,23 and elevates [Ca2þ]i to over 1 mM ex vivo.25 Therefore, one could expect cytosolic levels of Ca2þ in TM neurons to reach 1 mM during wakefulness and drop to the baseline near 60 nM during sleep.25 Accordingly, intermediate firing frequencies (i.e. 1e2 Hz) would be expected to correspond to intermediate phases of sleepewakefulness (e.g. non-REM sleep) and generated intermediate levels of [Ca2þ]i in the range of w300 nM (Figure 5.3(C)). The observed changes in [Ca2þ]i were rapid and fully reversible. These results demonstrate a prolonged presence of high levels of [Ca2þ]i (i.e. >300 nM) in TM neurons during phases of activity that could correspond to TM firing during wakefulness. These high levels of [Ca2þ]i may be essential for controlling histamine release, a Ca2þ-dependent process, while several sources of Ca2þ may contribute to this regulation, including high-threshold voltageactivated Ca2þ ion channels (HVACCs),25 Ca2þ stores (Figure 5.3), and NaþeCa2þ exchangers.38 Interestingly, transient elevations of Ca2þ levels elicited by Ca2þ release from cytosolic stores inhibited spontaneous firing of TM neurons and, thus, would be expected to inhibit histamine release due to activation of Ca2þdependent Kþ ion channels (KCa) (Figure 5.3(D)e(E)). These results suggest intimate links between Ca2þ stores and KCa within the TM cytoplasm and the ability of Ca2þ stores to modulate neuronal excitability of TM neurons. Given the role of TM neurons in circadian rhythms regulation, Ca2þ stores may participate in the maintenance of the sleepewakefulness cycle. The involvement of KCa in the inhibition of TM spontaneous firing elicited by Ca2þ release from internal stores is supported by the sensitivity of caffeinemediated responses to the external concentration of Kþ ions ([Kþ]o) (Figure 5.4) and ryanodine (Ry) (an
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irreversible agonist of Ry receptors, RyRs) (Figure 5.3(F)e(G)), as well as 50 mM cyclopiazonic acid (CPA, a blocker of Ca2þ-ATPase) (not shown). These results are summarized in Figures 5.3(H) and 5.4. The presence of intimate links between Ca2þ stores and KCa is supported by the similarity in the rates of onsets of [Ca2þ]i elevation and KCa current (Figure 5.3(H), insert). However, the duration of elevated [Ca2þ]i is clearly extended past the duration of KCa current, pointing to limitations in the potency of Ca2þ release for inhibition of TM firing. The presented data argue that elevation in TM [Ca2þ]i can significantly affect the firing patterns and behavior of TM neurons and, thus, modulate histamine release. However, although stimulation of Ca2þ stores can produce robust effects on TM firing (Figure 5.3(D)e(E)), the membrane voltage is likely to be the main player in integration of cytosolic Ca2þ signals in TM neurons25,37 as Ca2þ currents can be activated during afterhyperpolaryzation (AHP) with a strong dependence of the Ca2þ net charge on the membrane voltage where an equilibrium between activation and inactivation of multiple types of Ca2þ currents and AHP currents near the threshold of firing may support the physiological need in histamine. The HVACCs are expected to be especially important for regulation of spontaneous firing and histamine metabolism in the TM because of their noninactivating nature and control over the amount of Ca2þ influx during the action potential.35,37
STOCHASTIC ENHANCEMENT OF NEURONAL EXCITABILITY: CAN A SINGLE ION CHANNEL EXCITE THE ENTIRE NEURON? Nicotinic a7 acetylcholine receptors (a7 nAChRs) are widely expressed in the central nervous system. Under normal physiological conditions, the duration of a7 channel openings are extremely short (w0.1 ms39), but in the presence of type II positive allosteric modulators (a7-PAMs) such as PNU-120596, the duration of a7 open channels can be substantially increased (w1 s40). In the absence of PNU-120596, the generation of whole-cell a7 responses requires high synchrony of
voltage-clamp patch-clamp recordings (F) and the corresponding elevation in [Ca2þ]i (G) are blocked by 50 mM ryanodine (Ry) (FeG) added to Artificial Cerebrospinal Fluid (ACSF), supporting the involvement of Ca2þ stores and KCa. Traces shown in (F) and (G) share the same time scale shown in (G). (H) A summary of results obtained from n ¼ 9 TM neurons. Significance is defined by the p-value evaluated using an unpaired two-tail Student test. The insert illustrates the kinetics of KCa outward current versus elevation in [Ca2þ]i obtained from the same pressure application of 50 mM caffeine (Caf) to a TM neuron as shown in (FeG). Although the rates of onset of both processes are similar, the duration of elevated [Ca2þ]i is clearly extended past the duration of KCa current. (I) A simplified model of a TM neuron illustrating functional links between Ca2þ stores and KCa channels. Although normal TM spontaneous firing can elevate [Ca2þ]i to w1 mM, it is the release of Ca2þ from Ca2þ stores that activates KCa channels and inhibits spontaneous firing supporting these links. Source: Panels (AeC) are from Uteshev and Knot, 2005,25 with permission from Elsevier. (For color version of this figure, the reader is referred to the online version of this book.)
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FIGURE 5.4 The currentevoltage relationship and sensitivity to [KD]o of caffeine-induced KCa currents. (AeB) Typical current traces obtained from TM neurons held at various membrane voltages (indicated in the center) by brief pressure application of 10 mM caffeine. ACSF contained either 3 mM (A) or 8 mM (B) Kþ. (C) The currentevoltage dependence obtained from n ¼ 5 TM neurons. A clear depolarizing shift in the reversal potential is seen, reflecting an increase in [Kþ]o. This shift and the responsiveness to caffeine support the involvement of Ca2þ-dependent Kþ ion channels triggered by Ca2þ release from Ca2þ stores.
activation of many a7 channels. For instance, given the mean a7 open channel duration in the absence of PNU-120596 (i.e. w0.1 ms), theoretically, up to 10,000 a7 channels can open each second without considerable overlay and thus not result in detectable whole-cell responses. By contrast, the same 10,000 channels opened for an average duration of w1 s in the presence of PNU120596 would be expected to generate a 50e80 nA response. This example illustrates a strong synchronizing action of PNU-120596 on a7 nAChR-mediated ion channel activity. In the presence of PNU-120596 and physiological levels of choline (i.e. w5e20 mM), a weak persistent level of a7 nAChR activation can be achieved41,42 that is sufficient for triggering action potentials, thus enhancing the excitation of CA1 pyramidal neurons.43 The physiological importance of these effects at physiological temperatures has been recently questioned due to observations that the responsiveness of heterologously expressed a7 nAChRs to nicotinic agonists in the presence of PNU-120596 is significantly reduced at nearphysiological temperatures.44 However, PNU-120596 is effective in vivo (thus at physiological temperatures),45e49 and thus, although activation of a7 nAChRs in the presence of PNU-120596 is reduced at physiological temperatures, it can still be therapeutically beneficial. Alternatively, only the agonist binding and
activation of a7 nAChRs, but not ionic influx and activation of a7 nAChR-mediated ion channels, may be neuroprotective. In that event, a decrease in a7mediated ionic influx at physiological temperatures may in fact be neuroprotective in at least two ways: by preserving agonist-receptor binding (which may be directly neuroprotective) and reducing ionic influx (which may be directly neurotoxic). At least at room temperatures, multiple simultaneous openings of a7 channels may alter neuronal behavior. This is illustrated in Figure 5.5, where synchronous openings of at least four a7 ion channels send hippocampal CA1 interneurons to prolonged bursts of action potentials, sometimes lasting for over a minute. CA1 interneurons may directly inhibit CA1 pyramidal neurons via GABAergic synaptic inputs,50 or they may excite CA1 pyramidal neurons by inhibiting other CA1 interneurons (i.e. via disinhibition).51 Although PNU120596 would be expected to enhance activation of a7 nAChRs in pyramidal neurons and interneurons proportionally, the net effect of this activation remains unknown and is likely to be concentration dependent. Moreover, in the presence of PNU-120596, the activation of a7 nAChRs by ACh would be expected to be substantially enhanced, while the activation of non-a7 nAChRs should remain unchanged. Therefore, in the presence of PNU-120596, the net effect on the hippocampal output
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REFERENCES
the resistance of hippocampal principal neurons and interneurons to injury and various insults.42
CONCLUSION
FIGURE 5.5 Excitability of hippocampal CA1 interneurons can be enhanced by stochastic summation of a7 single ion channel openings in the presence of physiological choline and PNU-120596. A weak persistent activation of individual a7 nAChRs in hippocampal CA1 interneurons can be achieved by adding 10 mM choline and 1 mM PNU-120596 to ACSF. These a7 nAChR-mediated single-channel openings can be observed in whole-cell current-clamp recordings as step-like voltage deviations. A typical example of current-clamp recordings from a hippocampal CA1 interneuron is shown where simultaneous activation of four or more a7 nAChRs triggers prolonged bursts of action potentials. Recordings were done at room temperature. This enhanced excitability of inhibitory GABAergic interneurons would be expected to cause a prolonged inhibition of CA1 pyramidal neurons and, thus, the hippocampal output. A typical example of PNU-120596-induced bursts of action potentials recorded in whole-cell current-clamp mode is amplified and shown in the insert. Horizontal dashed lines indicate amplitudes of step-like voltage deviations that correspond to individual a7 single ion channel openings. A solid horizontal bar in front of traces indicates the membrane voltage of 50 mV.
of activation of CA1 a7 nAChRs will likely depend on the strength, timing, and location of cholinergic terminals and the relative densities of expression of preand postsynaptic a7 and non-a7 subtypes of nAChRs, as discussed.51,52 Nevertheless, since CA1 GABAergic interneurons act as the prime inhibitory source for CA1 pyramidal neurons, it is likely that enhanced bursting activity evoked by simultaneous activation of several a7 ion channels in the presence of PNU-120596 and physiological choline will inhibit some pyramidal neurons while disinhibiting others and, thus, alter the hippocampal output.51,52 Therefore, additional research is essential to determine whether an optimal range of a7 activation exists in the hippocampus that would allow preserving the hippocampal output while enhancing
This chapter illustrates the effectiveness of ex vivo techniques in supplementing in vivo approaches and providing a detailed understanding of ion channel and neurotransmitter receptor mechanisms in some network functions. Properties of individual neurons and synaptic connections shape the outputs of neuronal networks that are often synchronized and amplified by sensory inputs and their derivatives. Neuronal networks that are able to interpret patterns of sensory inputs are referred to as cognitive to distinguish them from autonomic networks that relay sensory inputs to higher brain regions or generate uninterpreted (i.e. unconscious) motor responses. However, there is a possibility that the interpretation of sensory information itself is input driven and may not be sustainable in the absence of a continuous sensory drive or its derivatives. In that event, the corresponding cognitive network signaling can be viewed as a sensory-driven reflex that initiates and supports its own interpretation by the network. This view supports a notion that links between cognitive and autonomic homeostases may be more intimate than currently appreciated.
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endogenous nicotinic agonists in cerebral ischemia. Br J Pharmacol. 2013; In Press. doi: 10.1111/bph.12247 (PMID: 23713819). 49. McLean SL, Idris N, Grayson B, et al. PNU-120596, a positive allosteric modulator of a7 nicotinic acetylcholine receptors, reverses a sub-chronic phencyclidine-induced cognitive deficit in the attentional set-shifting task in female rats. J Psychopharmacol. 2012; 26(9):1265e70. 50. Alkondon M, Pereira EF, Barbosa CT, Albuquerque EX. Neuronal nicotinic acetylcholine receptor activation modulates
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gamma-aminobutyric acid release from CA1 neurons of rat hippocampal slices. J Pharmacol Exp Ther. 1997;283(3): 1396e1411. 51. Ji D, Dani JA. Inhibition and disinhibition of pyramidal neurons by activation of nicotinic receptors on hippocampal interneurons. J Neurophysiol. 2000;83(5):2682e2690. 52. Ji D, Lape R, Dani JA. Timing and location of nicotinic activity enhances or depresses hippocampal synaptic plasticity. Neuron. 2001;31(1):131e141.
5 Network Experimental Approaches: Ex vivo Recording Victor V. Uteshev Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA
INTRODUCTION
DIRECT SYNCHRONIZATION VIA CHEMICAL AND ELECTRICAL SYNAPSES IN THE OLFACTORY BULB
In a functional brain, contributions of individual neurons can often be most effective if the neuronal activity is synchronized. Synchronization can be achieved either by a direct communication among neurons or by inhibition of asynchrony (i.e. reducing the chances of individual neurons to act differently from the rest of the group). For example, in the olfactory bulb, a direct communication among neurons via synaptic connections and gap junctions1 prevents external tufted (ET) cells of the same glomerulus from behaving asynchronously with the rest of the neuronal population, while natural pacemaker neurons of the hypothalamic histaminergic tuberomammillary (TM) nucleus that are electrically independent from one another (as discussed in this chapter) can be synchronously shut off during sleep by GABAergic inputs from the ventrolateral preoptic nucleus.2 Therefore, in some cases, the impact of individual neurons on the network status can be deduced either from understanding of the network structure as well as synaptic and/or gap junction connections, or from knowledge of pharmacology and kinetics of the external inputs and neuronal receptors involved in signaling. Neurons, on the other hand, may define the level of sustained excitability of neuronal networks, network stability, and sensitivity to the action of endogenous and exogenous compounds. Although “in vitro” is a term that has been often applied to brain slice work, it is perhaps more accurate to label acute slice work as “ex vivo”, which is done in this chapter. The term “in vitro” is used to refer to cultured neurons and cell lines.
Neuronal Networks in Brain Function, CNS Disorders, and Therapeutics http://dx.doi.org/10.1016/B978-0-12-415804-7.00005-8
The olfactory bulb is a complex and intriguing brain structure involved in encoding, transfer, processing, and decoding of odorant-evoked sensory information. It has a multifaceted network of interneuronal connections that include dendrodendritic chemical synapses and electrical synapses, also known as gap junctions.3 Organization and properties of neuronal and synaptic connections facilitate neuronal synchronization, which may be critical for effective odor discrimination.4e6 Moreover, optimal neuronal synchronization may enhance transmission of information from sensory receptors to higher order brain structures.7,8 Integration and processing of sensory olfactory information at the level of the olfactory bulb involve stimulation of a complex neuronal circuitry, specific patterns of activation of chemical and electrical synapses, and coordination of activity of multiple glomeruli. The glomeruli of the olfactory bulb serve as the sites of the first synapse for olfactory sensory inputs, which are processed by three types of glomerular juxtaglomerular (JG) neurons: ET, periglomerular (PG), and short axon (SA) cells. Olfactory bulb output neurons exhibit highly synchronized activity in acute ex vivo preparation of brain slices despite deprivation of their normal sensory inputs. Synchronous activity is common among output neurons whose tufted dendrites belong to the same glomerulus. Such synchronization was found among mitral cells,9 ET cells,1 tufted cells,10 and mitral cells.11 Furthermore, additional interactions among neurons within the same glomerulus or different glomeruli arise from
67 Copyright Ó 2014 Elsevier Inc. All rights reserved.
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FIGURE 5.1 Synchronous slow and fast Excitatory Post-Synaptic Currents (EPSCs) in ET cells of the same glomerulus. All data presented in this figure were obtained from the same ET cell pair. (A) Simultaneous extracellular recordings from two ET cells with correlated spike bursting (correlated spikes shown in the inset at right). (B) The same two cells were then recorded in whole-cell configuration in voltage clamp (HP of 60 mV) using electrodes (containing CsMeSO3 and QX-314). Note the synchronous slow (horizontal lines with arrows on both sides) and fast (asterisks; example shown at right) EPSCs. (C) Cross-correlogram of the spike trains (5 min recording sample, 1 ms bins) shows a significant correlation with a peak near zero lag time (see inset). (D) The cross-correlogram of the membrane current (50 s of intracellular recording sample, 2 ms bins) shows both a broad (small lines with arrows on both sides) and narrow (asterisk; see inset) correlation that peaked at zero time lag. The gray trace is the cross-correlogram of the membrane current traces after shifting the second trace by 5 s (to determine significance, see “Materials and Methods” in Ref. 1). (E) Cross-correlogram of the EPSC trains (5 min recording sample, 1 ms bins) shows a significant peak only in the bin at zero time lag, indicating synchronous EPSCs in the two recorded cells. The vertical axes in (CeE) demonstrate a cross-correlation value, C. (F) Cumulative probability histograms of the EPSC amplitudes of synchronous and asynchronous EPSCs recorded in both cells (during 5 min). The synchronous EPSCs exhibited significantly larger amplitude than asynchronous EPSCs (p < 0.0001, KeS test). (G) Scatterplot of the amplitude of synchronous EPSCs in cell 2 versus cell 1 and a linear regression fit showing a significant positive correlation. (H) Photograph of the two recorded cells after biocytin staining showing the overlap of dendrites in the same glomerulus (dashed line). Source: From Hayar et al., 2005,1 with permission from the Society for Neuroscience.
extensive lateral dendrites found in output neurons that establish dendrodendritic connections with interneurons.12 For example, a highly correlated activity was found among ET cells, SA cells, and PG cells13,14 and among mitral cells and granule cells.15 Using an ex vivo olfactory bulb slice preparation and dual patch-clamp recordings from the JG circuitry (Figure 5.1), it has been determined that ET cells spontaneously fire rhythmic spike bursts in the theta frequency
range and receive monosynaptic olfactory nerve input.1,16 By contrast, SA and most PG cells do not receive monosynaptic olfactory input, but receive instead monosynaptic excitatory inputs from ET cells. These observations demonstrated that ET cells serve as a major excitatory link between olfactory nerve input and other JG cells. Intriguingly, within the same glomerulus, spontaneous bursts among ET cells are highly correlated even in the absence of activity of fast chemical
INHIBITION OF ASYNCHRONY IN SPONTANEOUS ACTIVITY OF HYPOTHALAMIC HISTAMINERGIC TM NEURONS
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FIGURE 5.2 Simultaneous recordings from pairs of ventral TM (VTM) neurons in acute hypothalamic brain slices. Neighboring TM neurons (arrows) located within 200 mm from one another (A) were selected for simultaneous recordings of spontaneous action potentials (APs) in current-clamp patch-clamp experiments (BeC). The results obtained from analyzing nine pairs of TM neurons demonstrated random uncorrelated patterns of spontaneous APs of neighboring TM neurons. In each pair, spontaneous APs fired in one TM neuron were not detectable in the other TM neuron inhibited by injection of small (10 pA) hyperpolarizing currents (C).
synapses (i.e. when activity of GABAergic and glutamatergic fast synapses is blocked).1 Analysis of experimental data indicated that the synchronized spontaneous bursting of ET cells within the same glomerulus is coordinated by synaptic transmission and gap junction coupling among ET cells (Figure 5.1). Therefore, the synchronous bursting of ET cells and, thus, synchronized odor-evoked glomerular output may function to amplify transient sensory inputs and coordinate glomerular outputs to ensure that the high fidelity of the specific odorant profile information is relayed to higher order brain structures. Moreover, the bursting activity of ET cells is readily entrained by patterns of odor-evoked sensory inputs at sniffing frequencies.13 These results suggest that amplification of the sensory input by ET cells may help enhance synchronization of the glomerular network tuned to the sniffing of the animal and, thus, enhance its functionality. The presence of highly multifaceted neuronal interactions in the olfactory bulb circuitry may reflect a need of olfactory bulb neurons to tune their activity to the animal’s respiratory cycle17e19.
INHIBITION OF ASYNCHRONY IN SPONTANEOUS ACTIVITY OF HYPOTHALAMIC HISTAMINERGIC TM NEURONS The TM nucleus of the posterior hypothalamus represents the sole source of brain histamine and contributes to regulation of normal cognition, sleepewakefulness cycles, food and water consumption, and energy
metabolism (see Chapter 21).20,21 Histamine, a “waking substance”, regulates sleep and wakefulness, while bloodebrain barrier permeable histamine H1 receptor antagonists cause sedation. TM neurons are typical, natural pacemakers and demonstrate spontaneous firing in the absence of synaptic inputs in vivo,22,23 in vitro,24e26 and after acute dissociation.27,28 The firing pattern of TM neurons as well as the histamine concentration in cerebrospinal fluid follow a circadian rhythm (see Chapter 14) and depend on the state of arousal.29,30 The highest frequency of TM neuronal spontaneous firing (6e8 Hz) corresponds to wakefulness in vivo. The activity of TM neurons decreases during nonerapid eye movement (non-REM) sleep and ceases completely during rapid eye movement (REM) sleep.22,23 Blocking spontaneous firing by hypothalamic perfusion with tetrodotoxin inhibits histamine release,31,32 which has been shown to require extracellular Ca2þ in vivo33 and in vitro.31,34 The presence of both inactivating and noninactivating voltage-gated calcium channels in TM neurons35 produces a dynamic equilibrium supportive of sustained spontaneous firing via associated Ca2þ signaling.36,37 In contrast to olfactory ET cells, TM neurons do not communicate with their TM neighbors via chemical or electrical synapses (Figure 5.2). Instead, TM neurons fire action potentials independently and within a relatively narrow range (1e6 Hz) of frequencies unless inhibited, for instance, via GABAergic inputs from the ventrolateral preoptic nucleus.2 A strong relationship between the frequency of spontaneous firing and the corresponding Ca2þ influx in TM neurons has been established in ex vivo experiments
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FIGURE 5.3 Correlation among neuronal properties and function. (AeB) A typical example of simultaneous current-clamp (A) and Ca2þ (B) recordings from the same TM neuron. Spontaneous firing of TM neurons (A) correlates with elevation in [Ca2þ]i (B) measured using Ca2þ Green-1 dye included into the intracellular pipette solution. A graphic relationship between the frequency of spontaneous firing and Ca2þ influx (in mM and as a ratio of F/F0) is shown in (C). Firing frequencies (1e4 Hz) that are observed in vivo during wakefulness correspond to [Ca2þ]i w 0.2e1 mM and may support histamine release. (DeE) Caffeine inhibits spontaneous firing of TM neurons (D) by stimulating Ca2þ release from Ca2þ stores (E) and activation of Ca2þ-dependent potassium conductance (KCa). Both an outward conductance measured in
STOCHASTIC ENHANCEMENT OF NEURONAL EXCITABILITY: CAN A SINGLE ION CHANNEL EXCITE THE ENTIRE NEURON?
utilizing simultaneous Ca2þ imaging and electrophysiological patch-clamp recordings from TM neurons in acute brains slices.25 As the firing pattern of TM neurons defines the rate of histamine release and arousal state in mammals,22,23,31,32 this correlation between spontaneous firing and [Ca2þ]i predicts the levels of cytosolic Ca2þ that correspond to specific behavioral states (e.g. sleep or wakefulness) (Figure 5.3(A)e(C)). In particular, the maximum firing frequency observed in these ex vivo experiments was w4 Hz.25 This frequency corresponds to wakefulness in vivo22,23 and elevates [Ca2þ]i to over 1 mM ex vivo.25 Therefore, one could expect cytosolic levels of Ca2þ in TM neurons to reach 1 mM during wakefulness and drop to the baseline near 60 nM during sleep.25 Accordingly, intermediate firing frequencies (i.e. 1e2 Hz) would be expected to correspond to intermediate phases of sleepewakefulness (e.g. non-REM sleep) and generated intermediate levels of [Ca2þ]i in the range of w300 nM (Figure 5.3(C)). The observed changes in [Ca2þ]i were rapid and fully reversible. These results demonstrate a prolonged presence of high levels of [Ca2þ]i (i.e. >300 nM) in TM neurons during phases of activity that could correspond to TM firing during wakefulness. These high levels of [Ca2þ]i may be essential for controlling histamine release, a Ca2þ-dependent process, while several sources of Ca2þ may contribute to this regulation, including high-threshold voltageactivated Ca2þ ion channels (HVACCs),25 Ca2þ stores (Figure 5.3), and NaþeCa2þ exchangers.38 Interestingly, transient elevations of Ca2þ levels elicited by Ca2þ release from cytosolic stores inhibited spontaneous firing of TM neurons and, thus, would be expected to inhibit histamine release due to activation of Ca2þdependent Kþ ion channels (KCa) (Figure 5.3(D)e(E)). These results suggest intimate links between Ca2þ stores and KCa within the TM cytoplasm and the ability of Ca2þ stores to modulate neuronal excitability of TM neurons. Given the role of TM neurons in circadian rhythms regulation, Ca2þ stores may participate in the maintenance of the sleepewakefulness cycle. The involvement of KCa in the inhibition of TM spontaneous firing elicited by Ca2þ release from internal stores is supported by the sensitivity of caffeinemediated responses to the external concentration of Kþ ions ([Kþ]o) (Figure 5.4) and ryanodine (Ry) (an
=
71
irreversible agonist of Ry receptors, RyRs) (Figure 5.3(F)e(G)), as well as 50 mM cyclopiazonic acid (CPA, a blocker of Ca2þ-ATPase) (not shown). These results are summarized in Figures 5.3(H) and 5.4. The presence of intimate links between Ca2þ stores and KCa is supported by the similarity in the rates of onsets of [Ca2þ]i elevation and KCa current (Figure 5.3(H), insert). However, the duration of elevated [Ca2þ]i is clearly extended past the duration of KCa current, pointing to limitations in the potency of Ca2þ release for inhibition of TM firing. The presented data argue that elevation in TM [Ca2þ]i can significantly affect the firing patterns and behavior of TM neurons and, thus, modulate histamine release. However, although stimulation of Ca2þ stores can produce robust effects on TM firing (Figure 5.3(D)e(E)), the membrane voltage is likely to be the main player in integration of cytosolic Ca2þ signals in TM neurons25,37 as Ca2þ currents can be activated during afterhyperpolaryzation (AHP) with a strong dependence of the Ca2þ net charge on the membrane voltage where an equilibrium between activation and inactivation of multiple types of Ca2þ currents and AHP currents near the threshold of firing may support the physiological need in histamine. The HVACCs are expected to be especially important for regulation of spontaneous firing and histamine metabolism in the TM because of their noninactivating nature and control over the amount of Ca2þ influx during the action potential.35,37
STOCHASTIC ENHANCEMENT OF NEURONAL EXCITABILITY: CAN A SINGLE ION CHANNEL EXCITE THE ENTIRE NEURON? Nicotinic a7 acetylcholine receptors (a7 nAChRs) are widely expressed in the central nervous system. Under normal physiological conditions, the duration of a7 channel openings are extremely short (w0.1 ms39), but in the presence of type II positive allosteric modulators (a7-PAMs) such as PNU-120596, the duration of a7 open channels can be substantially increased (w1 s40). In the absence of PNU-120596, the generation of whole-cell a7 responses requires high synchrony of
voltage-clamp patch-clamp recordings (F) and the corresponding elevation in [Ca2þ]i (G) are blocked by 50 mM ryanodine (Ry) (FeG) added to Artificial Cerebrospinal Fluid (ACSF), supporting the involvement of Ca2þ stores and KCa. Traces shown in (F) and (G) share the same time scale shown in (G). (H) A summary of results obtained from n ¼ 9 TM neurons. Significance is defined by the p-value evaluated using an unpaired two-tail Student test. The insert illustrates the kinetics of KCa outward current versus elevation in [Ca2þ]i obtained from the same pressure application of 50 mM caffeine (Caf) to a TM neuron as shown in (FeG). Although the rates of onset of both processes are similar, the duration of elevated [Ca2þ]i is clearly extended past the duration of KCa current. (I) A simplified model of a TM neuron illustrating functional links between Ca2þ stores and KCa channels. Although normal TM spontaneous firing can elevate [Ca2þ]i to w1 mM, it is the release of Ca2þ from Ca2þ stores that activates KCa channels and inhibits spontaneous firing supporting these links. Source: Panels (AeC) are from Uteshev and Knot, 2005,25 with permission from Elsevier. (For color version of this figure, the reader is referred to the online version of this book.)
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FIGURE 5.4 The currentevoltage relationship and sensitivity to [KD]o of caffeine-induced KCa currents. (AeB) Typical current traces obtained from TM neurons held at various membrane voltages (indicated in the center) by brief pressure application of 10 mM caffeine. ACSF contained either 3 mM (A) or 8 mM (B) Kþ. (C) The currentevoltage dependence obtained from n ¼ 5 TM neurons. A clear depolarizing shift in the reversal potential is seen, reflecting an increase in [Kþ]o. This shift and the responsiveness to caffeine support the involvement of Ca2þ-dependent Kþ ion channels triggered by Ca2þ release from Ca2þ stores.
activation of many a7 channels. For instance, given the mean a7 open channel duration in the absence of PNU-120596 (i.e. w0.1 ms), theoretically, up to 10,000 a7 channels can open each second without considerable overlay and thus not result in detectable whole-cell responses. By contrast, the same 10,000 channels opened for an average duration of w1 s in the presence of PNU120596 would be expected to generate a 50e80 nA response. This example illustrates a strong synchronizing action of PNU-120596 on a7 nAChR-mediated ion channel activity. In the presence of PNU-120596 and physiological levels of choline (i.e. w5e20 mM), a weak persistent level of a7 nAChR activation can be achieved41,42 that is sufficient for triggering action potentials, thus enhancing the excitation of CA1 pyramidal neurons.43 The physiological importance of these effects at physiological temperatures has been recently questioned due to observations that the responsiveness of heterologously expressed a7 nAChRs to nicotinic agonists in the presence of PNU-120596 is significantly reduced at nearphysiological temperatures.44 However, PNU-120596 is effective in vivo (thus at physiological temperatures),45e49 and thus, although activation of a7 nAChRs in the presence of PNU-120596 is reduced at physiological temperatures, it can still be therapeutically beneficial. Alternatively, only the agonist binding and
activation of a7 nAChRs, but not ionic influx and activation of a7 nAChR-mediated ion channels, may be neuroprotective. In that event, a decrease in a7mediated ionic influx at physiological temperatures may in fact be neuroprotective in at least two ways: by preserving agonist-receptor binding (which may be directly neuroprotective) and reducing ionic influx (which may be directly neurotoxic). At least at room temperatures, multiple simultaneous openings of a7 channels may alter neuronal behavior. This is illustrated in Figure 5.5, where synchronous openings of at least four a7 ion channels send hippocampal CA1 interneurons to prolonged bursts of action potentials, sometimes lasting for over a minute. CA1 interneurons may directly inhibit CA1 pyramidal neurons via GABAergic synaptic inputs,50 or they may excite CA1 pyramidal neurons by inhibiting other CA1 interneurons (i.e. via disinhibition).51 Although PNU120596 would be expected to enhance activation of a7 nAChRs in pyramidal neurons and interneurons proportionally, the net effect of this activation remains unknown and is likely to be concentration dependent. Moreover, in the presence of PNU-120596, the activation of a7 nAChRs by ACh would be expected to be substantially enhanced, while the activation of non-a7 nAChRs should remain unchanged. Therefore, in the presence of PNU-120596, the net effect on the hippocampal output
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REFERENCES
the resistance of hippocampal principal neurons and interneurons to injury and various insults.42
CONCLUSION
FIGURE 5.5 Excitability of hippocampal CA1 interneurons can be enhanced by stochastic summation of a7 single ion channel openings in the presence of physiological choline and PNU-120596. A weak persistent activation of individual a7 nAChRs in hippocampal CA1 interneurons can be achieved by adding 10 mM choline and 1 mM PNU-120596 to ACSF. These a7 nAChR-mediated single-channel openings can be observed in whole-cell current-clamp recordings as step-like voltage deviations. A typical example of current-clamp recordings from a hippocampal CA1 interneuron is shown where simultaneous activation of four or more a7 nAChRs triggers prolonged bursts of action potentials. Recordings were done at room temperature. This enhanced excitability of inhibitory GABAergic interneurons would be expected to cause a prolonged inhibition of CA1 pyramidal neurons and, thus, the hippocampal output. A typical example of PNU-120596-induced bursts of action potentials recorded in whole-cell current-clamp mode is amplified and shown in the insert. Horizontal dashed lines indicate amplitudes of step-like voltage deviations that correspond to individual a7 single ion channel openings. A solid horizontal bar in front of traces indicates the membrane voltage of 50 mV.
of activation of CA1 a7 nAChRs will likely depend on the strength, timing, and location of cholinergic terminals and the relative densities of expression of preand postsynaptic a7 and non-a7 subtypes of nAChRs, as discussed.51,52 Nevertheless, since CA1 GABAergic interneurons act as the prime inhibitory source for CA1 pyramidal neurons, it is likely that enhanced bursting activity evoked by simultaneous activation of several a7 ion channels in the presence of PNU-120596 and physiological choline will inhibit some pyramidal neurons while disinhibiting others and, thus, alter the hippocampal output.51,52 Therefore, additional research is essential to determine whether an optimal range of a7 activation exists in the hippocampus that would allow preserving the hippocampal output while enhancing
This chapter illustrates the effectiveness of ex vivo techniques in supplementing in vivo approaches and providing a detailed understanding of ion channel and neurotransmitter receptor mechanisms in some network functions. Properties of individual neurons and synaptic connections shape the outputs of neuronal networks that are often synchronized and amplified by sensory inputs and their derivatives. Neuronal networks that are able to interpret patterns of sensory inputs are referred to as cognitive to distinguish them from autonomic networks that relay sensory inputs to higher brain regions or generate uninterpreted (i.e. unconscious) motor responses. However, there is a possibility that the interpretation of sensory information itself is input driven and may not be sustainable in the absence of a continuous sensory drive or its derivatives. In that event, the corresponding cognitive network signaling can be viewed as a sensory-driven reflex that initiates and supports its own interpretation by the network. This view supports a notion that links between cognitive and autonomic homeostases may be more intimate than currently appreciated.
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