A comparison of extracellular and intracellular recordings from medial septum/diagonal band neurons in vitro

doi:10.1016/0306-4522(91)90388-5

Neuroscience

Volume 42, Issue 2, 1991, Pages 451-462

https://doi.org/10.1016/0306-4522(91)90388-5 Get rights and content

Abstract

Firing patterns, action potential characteristics and some active membrane properties of guinea-pig medial septum/diagonal band neurons were studied in an in vitro slice preparation. A comparison was made between several types of cells classified according to either extracellularly recorded (n = 130) or intracellularly recorded (n = 30) electrophysiological characteristics. Using multi-barrel extracellular electrodes, three principal cell types were distinguished: slow rhythmic firing cells (29%), fast rhythmic firing cells (65%) and burst-firing cells (6%). Most slow firing cells could also be distinguished from other cell types by their relatively longer action potential duration and a characteristic cadmium-sensitive “hump” in the repolarization phase of the action potential. These characteristics of slow firing cells matched well with the characteristics of cholinergic, slow afterhyperpolarization cells previously identified with intracellular recordings. The action potential shape, firing rate and firing pattern characteristics of about 60% of extracellularly recorded fast rhythmic firing cells matched those of previously identified non-cholinergic fast afterhyperpolarization cells. The remaining extracellularly recorded, rhythmic firing cells (about 10% of slow firing and 40% of fast firing cells) had a mixture of characteristics which precluded unequivocal classification as to cholinergic or non-cholinergic cell type.

Using intracellular recording, the bee venom toxin, apamin, was shown to attenuate the characteristic post spike slow afterhyperpolarization of cholinergic cells and greatly enhanced their firing rate to depolarizing pulses. Apamin often attenuated a smaller and more transient afterhyperpolarization found in identified non-cholinergic cells, but firing rate was increased only slightly. Extracellular recordings from slow and fast rhythmic firing cells in the presence of apamin showed that excitability of slow firing cells was enhanced significantly more than fast firing cells. The apamin data support the hypothesis that extracellularly recorded slow firing cells are cholinergic.

We conclude that extracellularly recorded medial septum/diagonal band cells characterized by broad action potentials, slow rhythmic firing under microiontophoresed glutamate and a signature “hump” in the falling phase of the action potential are cholinergic cells. Extracellularly recorded fast rhythmic firing cells with a narrow action potential and no “hump” in the action potential are likely to be non-cholinergic cells. This extracellular electrophysiological “fingerprint” for cholinergic medial septum/diagonal band cells in vitro may now be extended to studies in vivo where controversy remains as to the neurochemical identity of basal forebrain cells involved in control of hippocampal slow rhythmic activity.

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We have recorded, for the first time, in non-anesthetized, head-restrained mice, a total of 339 single units in and around the laterodorsal (LDT) and sublaterodorsal (SubLDT) tegmental nuclei, which are located, respectively, in, or beneath, the periaqueductal gray and contain cholinergic neurons. The recordings were made during the complete wake–sleep cycle including wakefulness (W), slow-wave sleep (SWS), and paradoxical (or rapid eye movement) sleep (PS). The tegmental neurons displayed either a biphasic narrow or triphasic broad action potential. Seventy-six LDT or SubLDT neurons characterized by their triphasic long-duration action potentials were judged to be cholinergic and this was verified in anesthetized mice using neurobiotin juxtacellular labeling combined with choline acetyltransferase immunohistochemistry of the recorded cell. The 76 presumed cholinergic neurons discharged tonically at the highest rate during W and PS (W/PS-active neurons) as either single isolated spikes or clusters of two to five spikes, and 26 of them discharged selectively during W and PS, these W/PS-selective neurons being found mainly in the SubLDT. The clustering discharge was particularly prominent during PS, when it was associated with an obvious phasic change in the cortical electroencephalogram (EEG), and during waking periods, when it was accompanied by abrupt body movements. During the transition from sleep to waking, the cholinergic W/PS-selective neurons and the LDT or SubLDT noncholinergic W-selective neurons showed firing before the onset of W, while, at the transition from waking to sleep, they ceased firing before sleep onset. At the transition from SWS to PS, all the cholinergic neurons exhibited a significant increase in discharge rate before the onset of PS. The present study in mice supports the view that cholinergic and noncholinergic LDT and SubLDT neurons play an important role in tonic and phasic processes of arousal and cortical EEG activation occurring during W or PS, as well as in the sleep/waking switch.
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Correlation between firing behavior and K+ currents has previously been explored in the MS/DB. For example, apamin-sensitive Ca2+-dependent K+ currents underlying slow afterhyperpolarizing potentials (sAHP) have been described in slow firing cholinergic neurons of the MS/DB, and they are thought to contribute to the neurons’ strong spike adaptation (Griffith, 1988; Griffith and Matthews, 1986; Matthews and Lee, 1991). In contrast, fast-spiking neurons exhibiting markedly fast afterhyperpolarizing potential (fAHP) and little accommodation express modest apamin-sensitive Ca2+-dependent K+ current (Morris et al., 1999; Sotty et al., 2003).
The medial septum/diagonal band complex (MSDB) controls hippocampal excitability, rhythms and plastic processes. Medial septal neuronal populations display heterogeneous firing patterns. In addition, some of these populations degenerate during age-related disorders (e.g. cholinergic neurons). Thus, it is particularly important to examine the intrinsic properties of theses neurons in order to create new agents that effectively modulate hippocampal excitability and enhance memory processes. Here, we have examined the properties of voltage-gated, K⁺ currents in electrophysiologically-identified neurons. These neurons were taken from young rat brain slices containing the MS/DB complex. Whole-cell, patch recordings of outward currents were obtained from slow firing, fast-spiking, regular-firing and burst-firing neurons. Slow firing neurons showed depolarization-activated K⁺ current peaks and densities larger than in other neuronal subtypes. Slow firing total current exhibited an inactivating A-type current component that activates at subthreshold depolarization and was reliably blocked by high concentrations of 4-AP. In addition, slow firing neurons expressed a low-threshold delayed rectifier K⁺ current component with slow inactivation and intermediate sensitivity to tetraethylammonium. Fast-spiking neurons exhibited the smaller I_K and I_A current densities. Burst and regular firing neurons displayed an intermediate firing phenotype with I_K and I_A current densities that were larger than the ones observed in fast-spiking neurons but smaller than the ones observed in slow-firing neurons. In addition, the prevalence of each current differed among electrophysiological groups with slow firing and regular firing neurons expressing mostly I_A and fast spiking and bursting neurons exhibiting mostly delayer rectifier K⁺ currents with only minimal contributions of the I_A. The pharmacological or genetic modulations of these currents constitute an important target for the treatment of age-related disorders.
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The medial septum diagonal band complex (MS/DB) projects via cholinergic and GABAergic pathways to the hippocampus and plays a key role in the hippocampal theta rhythm. In the MS/DB we have previously described a population of fast spiking GABAergic neurons that contain parvalbumin and mediate theta frequency activity in vitro. The Kv3.1 potassium channel is a delayed rectifier channel that plays a major role in fast spiking neurons in the CNS, and has previously been localized in the MS/DB. To determine which cell types in the MS/DB express the Kv3.1b ion channel subunit, transgenic mice in which the expression of GABAergic and glutamate markers are associated with the expression of green fluorescent protein (GFP; GAD67-GFP and VGluT2-GFP mice, respectively) were used for immunofluorescence and axonal tract tracing. Electrophysiological studies were also carried out on rat MS/DB slices to examine the role of the Kv3.1 channel in theta frequency oscillations. The results for the MS/DB were as follows: (1) cholinergic cells did not express GFP in either GAD67-GFP or VGluT2-GFP mice, and there was GAD67 immunoreactivity in GFP-positive neurons in GAD67-GFP mice and in a small proportion (6%) of GFP-positive neurons in VGluT2-GFP mice. (2) Kv3.1b immunofluorescence was associated with the somata of GABAergic neurons, especially those that contained parvalbumin, and with a minority of glutamatergic neurons, but not with cholinergic neurons, and with GABAergic axonal terminal-like processes around certain GABAergic neurons. (3) Both Kv3.1b-positive and -negative GABAergic neurons were septo-hippocampal, and there was a minor projection to hippocampus from VGluT2-GFP neurons. (4) Kainate-induced theta oscillations in the MS/DB slice were potentiated rather than inhibited by the Kv3.1 blocker 4-aminopyridine, and this agent on its own produced theta frequency oscillations in MS/DB slices that were reduced by ionotropic glutamate and GABA receptor antagonists and abolished by low extracellular calcium. These studies confirm the presence of heterogeneous populations of septo-hippocampal neurons in the MS/DB, and suggest that presence of Kv3.1 in the GABAergic neurons does not contribute to theta activity through fast spiking properties, but possibly by the regulation of transmitter release from axonal terminals.
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In our study, we fail to correlate any emerging cluster subgroup with a particular morphological type. It is generally accepted that slow firing neurons express predominantly a cholinergic phenotype (Matthews and Lee, 1991; Jones et al., 1999). However, recently described septal glutamatergic neurons (Colom et al., 2005) may also express a slow firing phenotype (Sotty et al., 2003; Manseau et al., 2005).
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The data suggest an additional means by which neurons of different physiological and/or morphological categories might be identified. Extracellular spike waveforms have been used in many studies to differentiate cell types and to determine important functional differences between these cell types (Nisenbaum et al., 1988; Matthews and Lee, 1991; Rao et al., 1999; Gur et al., 1999; Bruno and Simons, 2002; Rauske et al., 2003). Cholinergic neurons in the medial septum/diagonal band exhibit a slow calcium-activated potassium conductance, which results in a prominent hump in extracellular recorded spikes, and a similar hump in the first derivative of intracellular spikes; this hump distinguishes these cells from non-cholinergic neurons in this region (Matthews and Lee, 1991).
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A comparison of extracellular and intracellular recordings from medial septum/diagonal band neurons in vitro

Abstract

Brain Res.

Neuroscience

Prog. Neurobiol.

Neurosci. Lett.

Neuroscience

Brain Res. Rev.

Brain Res. Rev.

Brain Res.

Neuroscience

Neurosci. Lett.

Neurobiol. Aging

Brain Res.

Brain Res. Rev.

Neuroscience

Life Sci.

Brain Res.

Neurochem. Int.

Brain Res. Bull.

Neurosci. Lett.