Activation of the mouse primary visual cortex by medial prefrontal subregion stimulation is not mediated by cholinergic basalo-cortical projections

Abstract

The medial prefrontal cortex (mPFC) exerts top-down control of primary visual cortex (V1) activity. As there is no direct neuronal projection from mPFC to V1, this functional connection may use an indirect route, i.e., via basalo-cortical cholinergic projections. The cholinergic projections to V1 originate from neurons in the horizontal limb of the diagonal band of Broca (HDB), which receive neuronal projections from the ventral part of the mPFC, composed of prelimbic (PrL) and infralimbic cortices (IL). Therefore, the objective of this study was to determine whether electrical stimulation of mice mPFC subregions activate (1) V1 neurons; and (2) HDB cholinergic neurons, suggesting that the HDB serves as a relay point in the mPFC-V1 interaction. Neuronal activation was quantified using c-Fos immunocytochemistry or thallium autometallography for each V1 layer using automated particle analysis tools and optical density measurement. Stimulation of IL and PrL induced significantly higher c-Fos expression or thallium labeling in layers II/III and V of V1 in the stimulated hemisphere only. A HDB cholinergic neuron-specific lesion by saporin administration reduced IL-induced c-Fos expression in layers II/III of V1 but not in layer V. However, there was no c-Fos expression or thallium labeling in the HDB neurons, suggesting that this area was not activated by IL stimulation. Stimulation of another mPFC subarea, the anterior cingulate cortex (AC), which is involved in attention and receives input from V1, activated neither V1 nor HDB. The present results indicate that IL and PrL, but not AC, stimulation activates V1 with the minor involvement of the HDB cholinergic projections. These results suggest a functional link between the ventral mPFC and V1, but this function is only marginally supported by HDB cholinergic neurons and may involve other brain regions.

Keywords: autometallography; basal forebrain; cholinergic neurons; immunocytochemistry; neuronal activity; prefrontal cortex; thallium; visual cortex.

Figure 1

Location of the stimulation sites in the mouse medial prefrontal cortex at +1.94, +1.78 and +1.70 mm anterior from Bregma. Black dots represent the stimulated sites for the three mPFC stimulated subregions. Three examples of cresyl violet-stained sections at the level of the implantation sites are shown. The dotted lines indicate the location of the inserted stimulation electrode. AC, anterior cingulate cortex; IL, infralimbic cortex; PrL, prelimbic cortex. Scale bar = 1 mm.

Figure 2

Microphotographs representative of the neuronal activation in V1 detected by c-Fos immunocytochemistry (A,C,E) or thallium autometallography (B,D,F). c-Fos is a transcription factor expressed in the nucleus when neurons are activated. Thallium autometallography alternatively relies on the proportional uptake of K⁺ ions with neuronal activity. Note that c-Fos immunoreactivity is detected primarily in the layers II/III (A) and V (E) of V1 whereas TlAMG staining is observed in all layers (B,D,F) with layer IV (D) showing communicating fibers in TlAMG but not c-Fos (C). Scale bar = 10 µm.

Figure 3

Neuronal activity of the primary visual cortex induced by stimulation of medial prefrontal cortex subregions measured by c-Fos immunoreactivity. (A–C): Microphotographs of c-Fos immunostaining in the primary visual cortex following electrical stimulation of IL (A), PrL (B) and AC (C) (right panels) compared to the non-stimulated hemisphere (left panels) at low and high (indents) magnification. Histograms of the number of c-Fos immunoreactive neurons (per 13 µm² area) in the layers II/III, IV and V of the primary visual cortex after medial prefrontal cortex subregions stimulation (far right) are shown. The expression of c-Fos in V1 is increased in layers II/III and V following IL and PrL stimulation, but not following the AC stimulation. AC, anterior cingulate cortex; IL, infralimbic cortex; PrL, prelimbic cortex. Error bars = Standard Deviation (SD). Scale bar = 500 µm (panels) and 50 µm (indents). ** = p < 0.01.

Figure 4

Basal forebrain cholinergic neurons do not express c-Fos or thallium staining induced by IL stimulation. (A–C) Microphotographs of ChAT:EGFP neurons (green) in the basal forebrain area with a high concentration of EGFP cells in the HDB (A), c-Fos immunostaining (B) and thallium autometallography (C). There was no staining of ChAT positive cells either by c-Fos (B, right panel) or thallium autometallography (C, right panel) in the basal forebrain of coronal brain sections at Bregma +0.62 mm following electrical stimulation of the IL (right panels) compared to the non-stimulated hemisphere (left panels). (D,E) Histograms of the number of c-Fos immunoreactive cells, note the near absence of c-Fos positive cells (D) or thallium stained cells (E) in the HDB and substantia innominata following stimulation of the IL, which suggest HDB and substantia innominata cholinergic neurons are not involved in V1 activation to vmPFC stimulation. HDB, Horizontal limb of the Diagonal band of Broca; LPO, Lateral Preoptic area; MPA, Medial Preoptic area; SI, Substantia Innominata; VP, Ventral Pallidum; Tu, Olfactory Tubercle. Delimitation of areas was designated in accordance with the atlas. Error bars = SD. Scale bar = 250 µm.

Figure 5

Effect of the specific lesion of the cholinergic neurons on IL-induced neuronal activation of the primary visual cortex. (A) ChAT-immunostained neurons in the HDB and surrounding areas in control (left panel) and saporin-lesioned mice (right panel). (B,C) ChAT-immunostained cholinergic fibers of the primary visual cortex in layer II/III (B), layer IV (C) and layer V (D) for controls (left panels) and saporin-injected mice (right panels). Note the absence of cholinergic neurons in HDB, VDB and MS regions as well as cholinergic fibers in V1 after saporin injection. (E) Histograms of the number of c-Fos-immunoreactive neurons (per 13 µm² area) in the layers II/III, IV and V of the primary visual cortex after IL stimulation in intact (red) or cholinergic lesioned (green) animals and in non-stimulated hemisphere (blue). The c-Fos expression in V1 was increased in the layers II/III and V following IL stimulation but the increase was attenuated in the layer II/III after lesion of cholinergic fibers. Scale bar (A) = 100 µm, (D) = 10 µm. Error bars = SD. * = p < 0.05, ** = p < 0.01.

Figure 6

Neuronal activity of the primary visual cortex measured by thallium autometallography. (A) Thallium staining in the primary visual cortex after infralimbic cortex stimulation (right panel) compared to non-stimulated hemisphere (left panel). (B–D) Details of thallium staining in V1 layers II/III (B), IV (C) and V (D) of non-stimulated (left panels) and stimulated hemispheres (right panels). Because Tl⁺ accumulation is correlated with increased activity, the darker neurons (and the background neurites) indicate stronger activity. Quantification of thallium-stained neurons (E) and mean optical density values (F) in primary visual cortex at −3.28 mm from Bregma following infralimbic cortex stimulation. High gray scale values correspond to low staining intensity. In the primary visual cortex, neuron bodies were strongly stained in layers II/III (B, right panel) and V (D, right panel) as well as pass-through fibers in layer IV (C, right panel) after stimulation of the infralimbic cortex. Scale bars = 10 µm. Error bars = SD. * = p < 0.05, ** = p < 0.01.

Figure 7

Schematic representations of possible pathways from vmPFC to V1. Microphotographs of putative IL-induced c-Fos-immunostaining in intermediate cortical areas with corresponding quantification of c-Fos neurons number (A) midcingulate cortex (B-0.26 mm); (B) primary somatosensory cortex (B-0.96 mm) and (C) posterior parietal cortex (B-1.82 mm). Possible communication pathways between these cortices and the vmPFC, HDB, and V1 are shown in (D). These cortice might be possible relays in the communication between vmPFC and V1. S1, sensory motor cortex; MC, mid cingulate cortex; PPC, posterior parietal cortex. Scale bar = 250 µm. Error bars = SD. ** = p < 0.01.