Spontaneous activity of neostriatal cholinergic interneurons in vitro

Abstract

Neostriatal cholinergic interneurons fire irregularly but tonically in vivo. The summation of relatively few depolarizing potentials and their temporal sequence are thought to underlie spike triggering and the irregularity of action potential timing, respectively. In these experiments we used whole-cell, cell-attached, and extracellular recording techniques to investigate the role of spontaneous synaptic inputs in the generation and patterning of action potentials in cholinergic interneurons in vitro. Cholinergic cells were spontaneously active in vitro at 25 +/- 1 degrees C during whole-cell recording from 2 to 3 week postnatal slices and at 35 +/- 2 degrees C during cell-attached and extracellular recording from 3 to 4 week postnatal slices. A range of firing frequencies and patterns was observed including regular, irregular, and burst firing. Blockade of AMPA and NMDA receptors altered neither the firing rate nor the pattern, and accordingly, voltage-clamp data revealed a very low incidence of spontaneous EPSCs. GABAA receptor antagonists were also ineffective in altering the spiking frequency or pattern owing to minimal inhibitory input in vitro. Functional excitatory and inhibitory inputs to cholinergic cells were disclosed after application of 4-aminopyridine (100 microM), indicating that these synapses are present but not active in vitro. Blockade of D1 or D2 dopamine receptors or muscarinic receptors also failed to influence tonic activity in cholinergic cells. Together these data indicate that cholinergic interneurons are endogenously active and generate action potentials in the absence of any synaptic input. Interspike interval histograms and autocorrelograms generated from unit recordings of cholinergic cells in vitro were indistinguishable from those of tonically active neurons recorded in vivo. Irregular spiking is therefore embedded in the mechanism responsible for endogenous activity.

Fig. 1.

Morphological and physiological identification of neostriatal cholinergic interneurons. A, Synthetic projection micrograph of a cholinergic neuron prepared from a 300-μm-thick whole mount is shown. The large soma and thick primary dendrites that branch to form fine-diameter secondary and higher order processes are characteristic of cholinergic interneurons. In this particular example, the axonal arborization gives rise to a dense plexus that innervates the area surrounding the soma and dendrites.B, During whole-cell recording, cholinergic cells are readily identified by their response to intracellular current injection. Injection of a negative current pulse produces an initial hyperpolarization followed by anI_h-dependent sag in the membrane potential. Depolarizing current induces regular spiking and results in a long-lasting afterhyperpolarization after cessation of current injection. C, In the absence of applied current, spontaneous regular spiking (rate = 2.87 Hz; CV = 0.157) was observed in this particular neuron. The membrane potential is indicated for the initial point of each trace in Band C, and the recording was made at 35 ± 2°C.

Fig. 2.

The spectrum of firing patterns observed during whole-cell recording at 25 ± 1°C in slices from 2 to 3 week postnatal rats. A–C, Cholinergic interneurons exhibited a continuum of firing patterns including regular (CV = 0.159), irregular (CV = 0.715), and burst (CV = 3.059) firing.D, Histogram of the mean firing rate illustrates that the majority of cholinergic cells are spontaneously active at 25 ± 1°C and exhibit a range (0.00–3.06 Hz) of firing rates.E, Plot of the relationship between the mean firing rate and the CV of the interspike intervals reveals that, in general, neurons firing with higher rates exhibit more regular spiking patterns. The CV was only calculated in neurons that fired at >0.33 Hz and was generated from a 1 min sample period.

Fig. 3.

Cholinergic cells exhibit a range of firing rates and patterns during cell-attached recordings at 35 ± 2°C from 3 to 4 week postnatal rats. A, B, Regularly spiking cells were readily identified during cell-attached recording and exhibited a narrow unimodal Gaussian distribution in the ISI histogram (bin width = 5 msec; 2 min sample). C,D, Irregularly spiking cells were recognized by the large variability in the ISI and gave rise to unimodal ISI histograms with a large variance (bin width = 10 msec; 2.5 min sample). The fluctuations in the baseline of the cell-attached voltage-clamptrace result from the opening of large conductance channels. E, F, Burst firing was characterized by the clustering of spikes and produced a very skewed ISI histogram, with the peak corresponding to the modal intraburst interval and variable, long-duration ISIs corresponding to the interburst intervals (bin = 25 msec; 4.5 min sample).G, Cholinergic cells exhibited a range (0.00–9.52 Hz) of firing rates with the majority of cells (69%) spiking at >0.2 Hz.H, The relationship between CV and firing rate shows a general trend for more rapidly firing cells to be more regular.

Fig. 4.

Spontaneous excitatory synaptic inputs are not responsible for the tonic firing of cholinergic interneurons in vitro. A, B, Cell-attached voltage-clamp recordings show that pharmacological blockade of AMPA and NMDA receptors had no obvious effect on the spike rate or pattern.C, D, Application of CNQX (20 μm) and CPP (50 μm) did not produce any change in the ISI histogram (bin width = 10 msec; 2 min sample), and accordingly the firing frequency and CV were unaltered. Control data are in C. E, F, Group data illustrate that spontaneous excitatory inputs do not have any detectable effect on the firing rate (E; control = 1.25 ± 0.98 Hz; CNQX + CPP = 1.46 ± 1.04 Hz;n = 17; p > 0.2) or CV (F; control = 0.703 ± 0.373; CNQX + CPP = 0.595 ± 0.334; n = 17;p > 0.2).

Fig. 5.

Cholinergic interneurons receive minimal tonic excitatory input in vitro. A, At a holding potential of −60 mV in the presence of BMI (30 μm) and APV (50 μm), very few spontaneous inward currents were detected. Application of 4-AP (100 μm) caused the appearance of many, fast EPSCs that were blocked by DNQX (40 μm). B, The rapid kinetics of the 4-AP–induced AMPA receptor–mediated EPSCs is shown using a faster sweep speed. C, D, Further confirmation that the 4-AP–induced EPSCs are mediated by AMPA receptors is provided by the voltage-dependent reversal of these events. E, Cholinergic cells do not receive detectable spontaneous NMDA receptor–mediated synaptic inputs at 0 mV in the presence of BMI (30 μm) and CNQX (20 μm). Addition of 4-AP (100 μm) produced many slow, inward currents, which were confirmed by blockade with CPP (50 μm) to be caused by activation of NMDA receptors.F, Superimposed 4-AP–induced NMDA receptor–mediated EPSCs are shown at a faster sweep speed to illustrate their slow kinetics. G, H, Further confirmation that the 4-AP–induced events are mediated by NMDA receptors is provided by their voltage dependence.

Fig. 6.

Irregular spiking of cholinergic cells is not attributable to spontaneous inhibitory inputs in vitro.A, B, No discernible alteration in the firing rate or pattern of this cholinergic cell occurs after blockade of GABA_A receptors with BIC (30 μm).C, D, Examination of the ISI histograms (bin width = 10 msec; 2 min sample) generated from data from which the spike trains were taken (control data in C) reveals that BIC (30 μm) produces no obvious effect on the spiking rate or pattern. E, F, Grouped data reveal that blockade of GABA_A receptors with BIC (30 μm; n = 4) or SR-95531 (30 μm; n = 4) does not produce a significant alteration in the firing frequency (E; control = 4.13 ± 1.64 Hz; BIC or SR-95531 = 3.35 ± 1.57 Hz; n = 8; p > 0.2) or CV (F; control = 0.237 ± 0.097; BIC or SR-95531 = 0.257 ± 0.150; n = 8;p > 0.2) of cholinergic cells.

Fig. 7.

Cholinergic interneurons receive minimal spontaneous inhibitory synaptic input in vitro.A, In the presence of DNQX (40 μm) plus APV (50 μm) at a holding potential of −10 mV, a few small-amplitude outward currents are detectable. Application of 4-AP induced a large increase in the amplitude and frequency of spontaneous IPSCs, and their sensitivity to BMI (10 μm) indicates that they are mediated by GABA_A receptors.B, Superimposed sweeps at a faster speed are shown to illustrate the kinetics of the 4-AP–induced IPSCs. C,D, The IPSCs are confirmed by their voltage-dependent reversal close to the chloride equilibrium potential to be caused by GABA_A receptor activation.

Fig. 8.

Extracellular single-unit recordings of cholinergic cells in vitro. The ISI histogram (B, E, H,K) and autocorrelogram (C,F, I, L) accompanying eachtrace (A, D,G, J) are shown. Bin width is 10 msec. A–C, Regularly spiking cholinergic cells give rise to a narrow, unimodal Gaussian distribution in the ISI histogram (12.5 min sample). The autocorrelogram (2.5 min sample) exhibits multiple, uniformly spaced peaks, owing to the stationarity and regularity of the spike train. D–F, Irregularly firing cholinergic cells exhibit relatively stationary ISIs interspersed with periods of more variable spike timing. The corresponding ISI histogram (15.5 min sample) is unimodal but skewed toward theright. The autocorrelogram (5 min sample) shows a single peak, caused by the increased likelihood of firing at the end of the afterhyperpolarization, and little additional structure.G–I, Cholinergic cells also exhibit bursting or clustered firing that was characterized most clearly by an obvious bimodal distribution in the ISI histogram (10 min sample). The autocorrelogram (10 min sample) exhibits two clear peaks, the first corresponding to the intraburst intervals and the second to the interburst intervals. J–L, Cholinergic cells that fired in a seemingly random manner were also encountered. The ISI histogram (28 min sample) displays a clear modal value but is very skewed, and examination of the autocorrelogram (10 min sample) reveals that, other than the decreased probability of spike generation during the afterhyperpolarization, there is no structure to the spiking pattern.