HCN channels segregate stimulation-evoked movement responses in neocortex and allow for coordinated forelimb movements in rodents

Abstract

Key points: The present study tested whether HCN channels contribute to the organization of motor cortex and to skilled motor behaviour during a forelimb reaching task. Experimental reductions in HCN channel signalling increase the representation of complex multiple forelimb movements in motor cortex as assessed by intracortical microstimulation. Global HCN1^KO mice exhibit reduced reaching accuracy and atypical movements during a single-pellet reaching task relative to wild-type controls. Acute pharmacological inhibition of HCN channels in forelimb motor cortex decreases reaching accuracy and increases atypical movements during forelimb reaching.

Abstract: The mechanisms by which distinct movements of a forelimb are generated from the same area of motor cortex have remained elusive. Here we examined a role for HCN channels, given their ability to alter synaptic integration, in the expression of forelimb movement responses during intracortical microstimulation (ICMS) and movements of the forelimb on a skilled reaching task. We used short-duration high-resolution ICMS to evoke forelimb movements following pharmacological (ZD7288), experimental (electrically induced cortical seizures) or genetic approaches that we confirmed with whole-cell patch clamp to substantially reduce I_h current. We observed significant increases in the number of multiple movement responses evoked at single sites in motor maps to all three experimental manipulations in rats or mice. Global HCN1 knockout mice were less successful and exhibited atypical movements on a skilled-motor learning task relative to wild-type controls. Furthermore, in reaching-proficient rats, reaching accuracy was reduced and forelimb movements were altered during infusion of ZD7288 within motor cortex. Thus, HCN channels play a critical role in the separation of overlapping movement responses and allow for successful reaching behaviours. These data provide a novel mechanism for the encoding of multiple movement responses within shared networks of motor cortex. This mechanism supports a viewpoint of primary motor cortex as a site of dynamic integration for behavioural output.

Keywords: intracortical microstimulation; motor cortex; reaching behaviour.

Figure 1. Representative image of ICMS forelimb movement responses presented as an overlay on rodent motor cortex

Under baseline conditions, the majority of sites within motor cortex respond to short‐train ICMS with single contralateral (primary) forelimb movement. ICMS responses at a small number of sites present as combinations of two or more contralateral forelimb/hindlimb movements or bilateral forelimb/hindlimb movements, which are collectively termed multiple movements. The bilateral forelimb movements include but are not exclusively mirror‐type movements. Maps are the appropriate size for 37–39 day old rats (Young et al. 2012). Dashed lines indicate multiple movement responses.

Figure 2. Spread of fluorescein dye across motor cortex following application at cortical surface (30 μl) or intracortical infusion (5 μl)

A, modified rat atlas from Paxinos & Watson (2006). Each section is labelled as distance from Bregma (mm). Scale bar = 2 mm. B, spread of fluorescein dye (green) following cortical surface application as performed for ZD7288 in ICMS experiments. Representative slices are from 30 min after application and were similar to the 15 and 45 min time‐points. C, spread of fluorescein dye following intracortical infusion as performed for ZD7288 in skilled reaching task experiments. Representative slices are from 45 min after infusion to match the duration of a single behavioural session. Vertical bands within fluorescence are artefacts of image acquisition.

Figure 3. Acute blockade of HCN channels with ZD7288 in motor cortex significantly increases expression of multiple movement responses in naïve rats

A, representative images of forelimb motor maps before (top row) or after (bottom row) surface application of aCSF (left; n = 6) or ZD7288 (right; n = 6). Line = Bregma; ‘A’ = anterior–posterior axis; ‘M’ = medial–lateral axis. B, per cent forelimb motor map area exhibiting multiple movement responses was significantly increased in post‐ZD7288 animals. C, mean primary forelimb map area was not altered by acute ZD7288 treatment. D, mean primary forelimb thresholds were not altered by acute ZD7288 treatment. E, under alpha chloralose, the normalized forelimb motor map area exhibiting multiple movement responses was also significantly increased in post‐ZD7288 animals (n = 9) whereas this was not observed in animals given aCSF (n = 8). F, under alpha chloralose, mean primary forelimb map area was not altered by acute ZD7288 treatment. G, under alpha chloralose, mean primary forelimb thresholds were reduced by acute ZD7288 treatment. ^* P < 0.05.

Figure 4. Repeated experimental seizures reduce I _h in L5PCs in motor cortex

A, representative responses of L5PCs from motor cortex to a series of hyperpolarizing voltage steps for Control (left; n = 14) and Seizure (right; n = 15) conditions (V _m = –50 mV). Recordings were made before (Top) and during (Middle) bath application of ZD7288 (30 μm); digital subtraction of the post‐drug response from the pre‐drug response is plotted below to identify ZD7288‐sensitive components. B, I _h responses (steady‐state amplitude of digitally subtracted traces) were significantly reduced in L5PCs from seizure animals. C, I _h tail current amplitudes were also significantly reduced in the Seizure condition. ^* P < 0.05.

Figure 5. Repeated experimental seizures increase expression of multiple movement responses in motor cortex

A, representative motor maps for Control (left; n = 10) and Seizure (right; n = 10) conditions. B, mean per cent of total forelimb motor map occupied by multiple movement responses was significantly increased after seizures. C, mean multiple movement thresholds were not significantly different between control and seizure conditions. D, mean primary forelimb map area was significantly increased in animals given seizures. E, mean primary forelimb thresholds were not altered by seizures. ^* P < 0.05.

Figure 6. HCN1^KO mice exhibit increased expression of multiple movement responses

ICMS maps were assessed in C57BL/6J mice pre/post‐aCSF (n = 4), C57BL/6J mice pre/post‐ZD7288 (n = 5) and HCN1^KO mice pre/post‐ZD7288 (n = 6). A, a significantly larger proportion of multiple movement responses was detected in HCN1^KO mice pre‐ZD7288 and C57BL/6J mice post‐ZD7288 relative to C57BL/6J mice either pre/post‐aCSF or pre‐ZD7288. B, mean multiple movement thresholds were significantly increased in C57BL/6J mice after ZD7288. C, mean primary forelimb map area was not altered by ZD7288. D, mean primary forelimb thresholds were significantly increased in C57BL/6J mice after ZD7288. ^* P < 0.05.

Figure 7. HCN1^KO mice exhibit deficits in reaching accuracy on a skilled reaching task

A, C57BL/6J (n = 4) and HCN1^KO (n = 6) mice did not significantly differ in the number of reach attempts. B, C57BL/6J mice achieved significantly greater per cent reaching success than HCN1^KO mice on several training days. C, mean ranked scores for reaching components. Significantly greater error scores representing atypical motor movements were observed in HCN1^KO mice on Digits to Midline and Elbow to Midline. ^* P < 0.05.

Figure 8. Acute pharmacological inhibition of HCN channels within motor cortex reduces reaching accuracy and increases atypical movements on a skilled reaching task

A, the number of reaching attempts was not significantly affected by intracortical saline (n = 6) or ZD7288 infusion (n = 6). A non‐significant increase in attempts with ZD7288 was noted. B, per cent reaching accuracy was reduced in animals given intracortical infusion of ZD7288 relative to baseline performance or vehicle infusion. Vehicle did not affect reaching accuracy C, mean ranked scores for reaching components. Significantly greater error scores representing atypical movements were observed during ZD7288 infusion relative to vehicle infusion on Advance and Grasp. ^* P < 0.05.