Deactivation of the inferior colliculus by cooling demonstrates intercollicular modulation of neuronal activity

Abstract

The auditory pathways coursing through the brainstem are organized bilaterally in mirror image about the midline and at several levels the two sides are interconnected. One of the most prominent points of interconnection is the commissure of the inferior colliculus (CoIC). Anatomical studies have revealed that these fibers make reciprocal connections which follow the tonotopic organization of the inferior colliculus (IC), and that the commissure contains both excitatory and, albeit fewer, inhibitory fibers. The role of these connections in sound processing is largely unknown. Here we describe a method to address this question in the anaesthetized guinea pig. We used a cryoloop placed on one IC to produce reversible deactivation while recording electrophysiological responses to sounds in both ICs. We recorded single units, multi-unit clusters and local field potentials (LFPs) before, during and after cooling. The degree and spread of cooling was measured with a thermocouple placed in the IC and other auditory structures. Cooling sufficient to eliminate firing was restricted to the IC contacted by the cryoloop. The temperature of other auditory brainstem structures, including the contralateral IC and the cochlea were minimally affected. Cooling below 20°C reduced or eliminated the firing of action potentials in frequency laminae at depths corresponding to characteristic frequencies up to ~8 kHz. Modulation of neural activity also occurred in the un-cooled IC with changes in single unit firing and LFPs. Components of LFPs signaling lemniscal afferent input to the IC showed little change in amplitude or latency with cooling, whereas the later components, which likely reflect inter- and intra-collicular processing, showed marked changes in form and amplitude. We conclude that the cryoloop is an effective method of selectively deactivating one IC in guinea pig, and demonstrate that auditory processing in the IC is strongly influenced by the other.

Keywords: auditory pathways; auditory processing; commissure; cooling inactivation; guinea pig; inferior colliculus; single unit.

Figure 1

Photograph of the cryoloop used to cool the IC held in a micromanipulator. Two 19 gauge needles soldered to each end of the stainless steel cryoloop tubing acted as the inlet and outlet ports for the coolant. The manipulator allowed precise placement of the loop in contact with the exposed IC. Inset: detail of the cryoloop. The thermocouple tip allows precise monitoring of the cryoloop temperature. The surface of the cryoloop was placed in contact with the dorso-rostral part of the exposed IC. Scale bars = 5 mm.

Figure 2

(A) Schematic coronal section through the IC showing placement of the cryoloop and temperatures measured in the IC with a needle thermocouple during cooling in the left (cooled) and right IC. These measurements were made after removal of the overlying cortex. For electrophysiological recording experiments the cortex over the right IC was left intact with the result that the temperature would be ~2°C warmer in the right IC (see text). (B) Mean ± SD of temperature measured in the lateral-most penetration of the left IC and three similar cases (filled circles, solid line). Open circles and dashed lines show temperature as a function of depth in guinea pig auditory cortex re-plotted from Coomber et al. (2011) for comparison.

Figure 3

(A) Temperature of the cryoloop (filled circles) and at 1-mm depth in the contralateral IC (open circles) during repeated cycles of cooling of different duration. The temperature in the contralateral IC falls only a few degrees below control temperature. (B) Temperature in the contralateral IC (open circles) is relatively stable as the duration of the cooling cycle is increased progressively to 30 min. (C) Cooling the IC resulted in a less than 2°C reduction in temperature in the ipsilateral cochlear nucleus (CN, open circles) and (D) in the ipsilateral cochlear duct (open circles).

Figure 4

(A) Near coronal section through the IC following an experiment. Aspiration of the cortex overlying the left IC and placement of the cryoloop did not produce any noticeable trauma to the tissue. Blood at the midline (black arrowhead) and bilaterally at the ventro-lateral edges of the tectum (red arrowheads) resulted from aspiration of the cortex. Scale bar = 1 mm. (B) Neurons near the dorsal surface of the IC contacted by the cryoloop show no sign of damage. (C) Neurons within the cooled IC have normal morphology and no signs of ischemia are present. Scale bar in (B) and (C) = 50 μm.

Figure 5

The responses (Median ± IQR) of 12 single units to multiple presentations of a pure tone at CF, 20 dB re threshold, during stepwise cooling of the IC. Units were recorded along the dorso-ventral axis of the IC from 361 μm (A) to 3368 μm (L) from the dorsal surface. Cooling into the range 10–20°C induced a significant (p < 0.001) reduction in firing rate in all 12 units. In 2 units (D and G) firing rate increased significantly re control (p < 0.001) prior to decreasing at lower temperatures.

Figure 6

(A–C) PSTHs and (D–F) FRAs during control, cool to 14.4°C, and recovery conditions, respectively for a unit with CF = 2.1 kHz. The unit had an on-sustained PSTH and a V-type FRA under control conditions. During cooling only an occasional spike near stimulus onset remained, but the frequency tuning was retained. On recovery the unit regained its original PSTH shape and firing as a function of frequency returned to control levels.

Figure 7

(A) Schematic representation of multi-unit recordings made at approximately 1 mm steps in an electrode penetration along the dorso-ventral axis of the IC showing depth from the dorsal surface (black text) and CF of the recorded cluster (red text). (B) PSTHs for activity recorded at the four locations shown in (A) during control, cool, and recovery. Cooling resulted in a notable reduction of spiking in the three most dorsal positions, but had less effect in the deepest position. (C) Ratio of firing rate in the cool and control conditions as a function of unit CF for single and multi-unit cluster recordings pooled across experiments. The reduction in firing with cooling was most evident for low CFs (dorsal locations), while neurons with higher CFs in more ventral locations were less affected. Regression line (black) and 95% confidence limits (blue).

Figure 8

Histograms showing the effect of cooling induced deactivation for single units grouped by CF. All units with a CF ≤ 8 kHz showed a statistically significant reduction in firing on cooling re their control (pre-cool) values, whereas units with CF >8 kHz did not show a statistically significant reduction in firing during cooling. In all cases firing rates recovered close to control values on rewarming.

Figure 9

(A) PSTH and (B) ISIH for a unit with a chopper response (CF = 1.1 kHz) that showed a change in firing rate and temporal pattern (mediated by an increase in ISI) during cooling of the contralateral IC. Both rate and ISI values recovered on re-warming to control temperature.

Figure 10

(A,B) PSTHs of a broad-onset unit (CF = 8.1 kHz) recorded in the IC contralateral to the cryoloop before, during and after cooling. The number of spikes elicited to stimulation of the contralateral ear (A) increased during cooling and returned to the control value on recovery. In response to binaural stimulation (B) the unit showed an EI characteristic, but firing similarly increased with cooling. (C) Five examples of action potentials recorded during each of the three stages of the paradigm; the morphology of the action potential did not change during cooling of the contralateral IC.

Figure 11

Scatter plot showing control firing rate versus firing rate during cooling for single neurons recorded in the IC while cooling the contralateral IC. Dashed line shows the line of identity.

Figure 12

Averaged local field potentials (100 sweeps) recorded in the IC in response to stimuli (1-ms duration tone pulses) presented at time zero to (A) the ear contralateral to the recorded IC, (B) the ipsilateral ear, and (C) binaural stimulation. Responses are shown for control conditions (black), during cooling (blue) and rewarming (gray) of the contralateral IC. Cooling has little effect on the first peak in the waveform, but changes the amplitude and time course of components occurring at and after 10 ms post stimulus onset.