In total, we obtained whole-cell recordings from 114 pretectal nuclear complex (PNC) neurons. Slices included the caudal part of the pretectum (Fig. ), cells were recorded from the NOT, the posterior pretectal nucleus (PPN), and the olivary pretectal nucleus (OPN). Depolarizing current injections induced various spike patterns, like bursting (Fig. ), non-adapting regular spiking (Fig. ), or irregular spiking (Fig. ). Usually, increasing the current amplitude also increased the firing rate, however, in about 31% (n = 35) of the cells, the firing rate showed a clear maximum in response to intermediate depolarizing current injections and decreased upon further depolarization (Fig. ). Input resistances ranged from 201.0 to 776.3 MΩ (mean 410 ± 166.5 MΩ), resting potentials varied between -41.0 and -74.3 mV (mean -54.6 ± 8.5 mV). All cells tested, irrespective of the response type, responded to OT stimulation.
Figure 1 Schematic view of stimulation and recording sites. The box in the reconstructed section from the right midbrain in the left panel indicates the position of the PNC shown at higher magnification in the right panel (dorsal is up, medial is to the left). (more ...)
Figure 2 Different types of responses in PNC neurons evoked by depolarizing current injections. A-C, Whole-cell recordings from three individual PNC cells that showed burst firing in response to depolarizing currents. While the firing rate of cells in A and B (more ...)
Characteristics of spontaneous activity
Of the cells recorded, 73 PNC neurons showed spontaneous firing at resting potential. Camera lucida reconstructions revealed that these cells were characterized by large fusiform cell bodies (diam. 15 μm and above) and multipolar dendritic trees that did not show any preference in their orientation. Whenever axons were also visible, they could be followed to leave the pretectal area in a ventro-lateral direction which indicates that these cells project to extrapretectal targets (arrowheads in Fig. ). Their dendritic morphologies, however, did not allow to distinguish spontaneously active cells from PNC neurons that were not spontaneously active.
Figure 3 Morphological and physiological characteristics of PNC neurons with spontaneous activity in vitro. A, B, Reconstruction drawings of two biocytin-filled PNC neurons, insets indicate the cells' position within the PNC. Horizontal lines mark the dorsal border (more ...)
Physiologically, all spontaneously active PNC neurons were characterized by a regular firing pattern when recorded at resting potential without any injected current (Fig. ). The firing rate at resting potential of spontaneously active PNC cells varied between 0.9 and 9.4 imp/s (mean 3.0 ± 2.1 imp/s). Depolarizing current injections induced tonic firing patterns with only marginal adaptation (Fig. ). Responses to hyperpolarizing current injections showed no sign of inward rectification. Furthermore, following cessation of hyperpolarizing current steps we never observed rebound spikes. Spontaneously active PNC cells on average had significantly higher input resistances (mean 454.1 ± 164.7 MΩ, p < 0.001), more positive resting potentials (mean -50.4 ± 7.0 mV, p < 0.001) and lower spike thresholds (mean -55.0 ± 3.96 mV, p < 0.001) than cells that did not show spontaneous activity (331.44 ± 137.1 MΩ, -58.4 ± 8.0 mV, and -40.66 ± 6.44 mV, respectively).
In order to characterize the spike adaptation behavior of spontaneously active PNC cells, the holding potentials were increased in 5 mV steps by appropriate current injections in all recorded cells. In response to these depolarization steps, cells showed tonic increases of their firing rate without any sign of firing rate adaptation (Fig. ). Also, no phasic firing rate increases were observed following the depolarizations.
Figure 4 Response to intracellular depolarization of spontaneously active PNC cells. Depolarizing current steps induce tonic firing increases in this spontaneously active PNC cell. No phasic component appears in the response to the depolarization step. This behavior (more ...)
As could be already derived from current injections, the firing rate was directly correlated with the membrane potential. Increasing the membrane potential by positive current injections increased the firing rate until a maximum level was reached that could not be exceeded by further depolarization (Fig. ). Consequently, when the firing rate is plotted against the membrane potential, the course of the resulting function is sigmoidal (Fig. ).
Figure 5 Correlation between firing rate and membrane potential in spontaneously active PNC cells. A, Incremental intracellular depolarization leads to increasing firing rates without changing the regular firing pattern. B, When the firing rate is plotted as a (more ...)
In order to get an impression about the regularity of the firing of spontaneously active PNC cells, interspike intervals (ISI) during maintained firing were analyzed in more detail (Fig. ). Thus, maintained firing was recorded over a 10 s period at different membrane potentials and ISI histograms were generated from the recorded activity. ISIs obtained from these recordings followed a narrow unimodal Gaussian distribution with only little variation (Fig. ). According to the correlation between the firing rate and the membrane potential, depolarization of the cells resulted in shifts of the maximum of the Gaussian distribution towards lower ISI values. Depolarization, however, did not change the shape of the distribution. The regularity of the maintained firing of spontaneously active PNC cells is also supported by autocorrelograms of the recorded spike trains (Fig. ). The appearance of multiple equally spaced peaks in the autocorrelogram results from the regular timing of single spikes.
Figure 6 Regularity of the firing pattern of spontaneously active PNC cells. A, Current clamp recording from a PNC cell at slightly depolarized membrane potential. B, The interspike interval histogram shows a narrow Gaussian distribution with only little variation (more ...)
Generation of spontaneous activity in vitro
In order to test whether the spontaneous activity of PNC neurons in vitro depends on excitatory input, we first suppressed glutamatergic synaptic transmission and pharmacologically blocked AMPA receptors in 13 spontaneously active PNC cells (Fig. ). As a control for the effectiveness of AMPA receptor blockade, the influence of the AMPA receptor antagonist CNQX on postsynaptic responses was monitored. In all cells tested, bath application of 20 μM CNQX resulted in a complete loss of EPSCs after electrical stimulation of optic tract afferent fibers (Fig. ). Although excitatory input was obviously blocked by CNQX application, the maintained firing remained unchanged (Fig. ). In particular, no drop in the firing rate was observed that could have been induced by a possible loss of excitatory input. Furthermore, the comparison of both the ISI distribution (Fig. ) and the autocorrelograms (Fig. ) obtained from spike trains before and during CNQX application did not show any significant difference. Hence, both the generation of spontaneous activity and its patterning seem to be independent from excitatory input via AMPA receptors. Similar results were achieved when NMDA receptors were blocked by bath application of 50 μM APV or when 2 mM kynurenic acid was applied to simultaneously block AMPA and NMDA receptors (N = 19).
Figure 7 Spontaneous activity of PNC is independent from tonic excitatory input. A single electric shock delivered to the optic tract lateral from the recorded neuron evokes a single peak EPSC (A) that completely disappears after AMPA receptor blockade by bath (more ...)
After having excluded glutamatergic synaptic inputs as a trigger for maintained firing, we tried to remove all synaptic input by adding cobalt to the extracellular medium in 12 spontaneously active PNC cells. This blocks the influx of calcium into the presynaptic terminal and thus prevents vesicular neurotransmitter release. Adding 1.5 mM CoCl2 to the bath completely suppressed all electrically evoked postsynaptic currents (Fig. ) in all cells tested. In contrast to the complete loss of postsynaptic currents, however, the maintained firing always remained unchanged (Fig. ). As during glutamate receptor blockade, no reduction of the firing rate was observed that could have indicated the removal of an excitatory input. In addition, no increase of the firing rate appeared that could have indicated a loss in tonic inhibitory input regulating maintained activity. Finally, examination of the ISI distribution in the spike trains demonstrated that the patterning of the maintained activity also did not show any significant difference in the presence of Cobalt (Fig. ). This indicates that spontaneously active PNC cells generate their firing intrinsically without any external synaptic input.
Figure 8 Tonic synaptic input does not contribute to the generation of spontaneous activity in PNC cells. Postsynaptic responses obtained by electrical optic tract stimulation (A) can also be blocked by bath application of 1.5 mM CoCl2 (D). However, the spontaneous (more ...)
In current-clamp mode, each action potential was preceded by a depolarizing ramp (see, for example, Figs. and ). When cells were hyperpolarized to membrane potentials just below their resting potential single depolarizing ramps appeared that were not followed by an action potential. Concomitantly, in voltage-clamp mode, each unclamped action potential was preceded by an depolarizing inward current (Fig. ). Because they did not disappear after substitution of calcium by cobalt in the external solution these current ramps were calcium independent. However, when 1 μM tetrodotoxin (TTX), a selective blocker of sodium channels, was added to the bath solution current ramps were eliminated together with the action potentials (Fig. ) in all seven cells tested.
Figure 9 Spontaneous activity in PNC cells depends on a sodium conductance. In control solution (A) this cell showed regular firing pattern with depolarizing inward currents preceding each action potential. When 1 μM TTX was added to the bath the firing (more ...)