The underside of the cerebral cortex: layer V/VI spiny inverted neurons

Abstract

This paper presents an account of past and current research on spiny inverted neurons--alternatively also known as 'inverted pyramidal neurons'--in rats, rabbits and cats. In our laboratory, we have studied these cells with a battery of techniques suited for light and electron microscopy, including Nissl staining, Golgi impregnation, dye intracellular filling and axon retrograde track-tracing. Our results show that spiny inverted neurons make up less than 8.5 and 5.5% of all cortical neurons in the primary and secondary rabbit visual cortex, respectively. Infragranular spiny inverted neurons constitute 15 and 8.5% of infragranular neurons in the same animal and areas. Spiny inverted neurons congregate at layers V-VI in all studied species. Studies have also revealed that spiny inverted neurons are excitatory neurons which furnish axons for various cortico-cortical, cortico-claustral and cortico-striatal projections, but not for non-telencephalic centres such as the lateral and medial geniculate nuclei, the colliculi or the pons. As a group, each subset of inverted cells contributing to a given projection is located below the pyramidal neurons whose axons furnish the same centre. Spiny inverted neurons are particularly conspicuous as a source of the backward cortico-cortical projection to primary visual cortex and from this to the claustrum. Indeed, they constitute up to 82% of the infragranular cells that furnish these projections. Spiny inverted neurons may be classified into three subtypes according to the point of origin of the axon on the cell: the somatic basal pole which faces the cortical outer surface, the somatic flank and the reverse apical dendrite. As seen with electron microscopy, the axon initial segments of these subtypes are distinct from one another, not only in length and thickness, but also in the number of received synaptic boutons. All of these anatomical features together may support a synaptic-input integration which is peculiar to spiny inverted neurons. In this way, two differently qualified streams of axonal output may coexist in a projection which arises from a particular infragranular point within a given cortical area; one stream would be furnished by the typical pyramidal neurons, whereas spiny inverted neurons would constitute the other source of distinct information flow.

Fig. 1

Camera-lucida drawings of three immature inverted neurons in layer VI of the rabbit temporal cortex. The cells were impregnated by the Golgi-Stensaas procedure on postnatal day 1. Axons are marked by the letter ‘a’. All of them arise from the main (apical) dendrite, which is orientated towards the white matter. Note the growth beads in dendrites and axons. These cells will most probably be spiny when mature. This kind of spiny inverted cell is also typically referred to as an ‘inverted pyramidal neuron’ in the literature. Scale bar: 15 µm. This bar is at the border of layer VI with the white matter.

Fig. 2

Infragranular inverted neurons in the rabbit and cat adult cerebral cortex. All of them are spiny inverted neurons, as defined in this paper, except for the cell on the right in (a). Notice that the axon arises from the apical dendrite in many cells. Notice also that some apical dendrites are bifurcated. (a) The cell on the left is a fully fledged spiny inverted neuron. Notice the dendrite spines. The cell on the right, which is bigger than its companion, is a mature aspiny neuron with inverse dendrite polarization, possibly a basket cell. Both neurons lie in layer V of adult cat area 18. Golgi impregnation. Scale bar: 20 µm. (b) Long-range projecting, inverted neuron of the layer V/VI border, adult rabbit secondary visual cortex. This fully fledged cell was retrogradely labelled after fluorogold injection into the ipsilateral primary visual cortex. Scale bar: 15 µm. (c) Two long-range projecting, mature inverted neurons retrogradely labelled with WGA-HRP in layer V of adult cat posterolateral lateral suprasylvian sulcus (area PLLS). The histological section was Nissl counterstained. Tracer injection was delivered into ipsilateral area 17. Scale bar: 20 µm. (d) Camera-lucida illustration of two Golgi-impregnated mature spiny inverted neurons in the adult cat primary visual cortex. Notice the forked apical dendrite of the cell on the left. Scale bar: 25 µm. In this and the next illustration, slim horizontal bars indicate the border of layer VI with the white matter. (e) Camera-lucida drawing of three Golgi-impregnated mature spiny inverted neurons in layer VI of the adult cat primary visual cortex. Scale bar: 20 µm. (f) Long-range projecting, spiny inverted neuron in layer VI of adult cat area 18. This mature cell was retrogradely labelled after an injection of rhodamine latex beads into ipsilateral area 17. Then, in a semi-fixed slice, the cell was filled with Lucifer Yellow delivered through a pipette inserted into the soma under microscopic control. The cell was spiny (not appreciable due to the low magnification). Scale bar: 15 µm.

Fig. 3

Camera-lucida drawing of the soma and main portions of the dendrites and axon of a Golgi-impregnated neuron with reverse dendrite polarization. This fully fledged neuron was found in layer VI of the adult rat primary visual cortex. This neuron is a good example of an aspiny inverted pyramidal neuron, which is presumably an interneuron of inhibitory nature. Notice the apical dendrite pointing towards the white matter. All dendrites were short and thin. No spine was observed on them. Arrow points to the axon. Scale bar: 20 µm.

Fig. 4

Photomicrograph of a mature spiny inverted neuron of layer V of adult cat area 18. The cell was labelled by axon retrograde transport of rhodamine latex beads injected into ipsilateral area 17. Then the cell was filled with Lucifer Yellow through a pipette (*) inserted into the cell soma. Notice the forked apical dendrite. Arrowhead shows dendrite spines, some of which are magnified in the inset. Double arrowhead points to the cell axon. The axon comes out from the apical dendrite, which is orientated towards the white matter (not seen). Scale bar: 10 µm.

Fig. 5

Camera-lucida drawings of the soma and main portions of dendrites and axons of three Golgi-impregnated, mature spiny inverted neurons of layer VI of adult rat visual cortex. Notice that the axons (arrows) arise either from the apical dendrite (cell on the left), the basal somatic surface (cell on the right) or the somatic lateral flank (cell in the middle). Percentages indicate the incidence of each cell subtype out of 28 Golgi-impregnated spiny inverted neurons in infragranular layers of rat visual and sensorimotor areas. As seen with subsequent electron microscopy, each axon acquired a myelin sheet during its trajectory towards the white matter or the pia (cell on the right). Scale bar: 20 µm.

Fig. 6

Camera-lucida drawings of two spiny inverted neurons with axons firstly going straight towards the pia and then turning around completely, to assume a downward trajectory to the white matter. The immature cell on the left was retrogradely labelled in layer VI of rabbit secondary visual cortex after a biocytin injection into the primary visual cortex at postnatal day 3. The fully fledged cell on the right is a Golgi-impregnated, spiny inverted neuron of adult rat secondary visual cortex. Both axons emerged from the basal pole of the soma. They are marked by arrowheads (left cell), or an arrow (cell on the right). Subsequent electron-microscopy analysis revealed that the axon initial segment of the axons of the Golgi-impregnated neuron on the right and of the neuron depicted to the right in Fig. 5 had similar ultrastructural features. Scale bars: 20 µm.

Fig. 8

Electron microphotograph of a large axon terminal bouton (indicated by arrow) making a Gray Type II symmetric contact with the axon initial segment of a biocytin-filled spiny inverted neuron of layer V of adult rat sensorimotor cortex. Notice the polymorphic vesicles in the terminal bouton. The postsynaptic membrane density is obscured because of biocytin filling the axon initial segment. The electron microscopy image was taken at 15 000×. This synapse exemplifies those observed with electron microscopy at the axon initial segment of 11 mature spiny inverted neurons of adult rat cortex.

Fig. 7

Photomicrographs of mature projection neurons, retrogradely labelled with cholera toxin, fraction b (CTb) after injections into the adult rabbit primary visual cortex, ipsilateral to the injections. Arrowheads point to distinctively labelled inverted neurons, though not every labelled inverted neuron is indicated in this way. All of these labelled inverted neurons are presumably spiny inverted neurons, as defined in this paper. All sections are coronal. M: medial. L: lateral. (a) Tracer injection into the primary visual cortex. Notice CTb diffusion within the grey matter. (b) Labelling at the medial sulcus between the primary visual cortex and the retrosplenial cortex. Notice the horizontal orientation of the labelled inverted neurons (inset). (c) The vertical line indicates the transition between the primary and secondary visual cortex. This was delimitated by observing Nissl-stained, consecutive rostral and caudal sections. The single asterisk marks a clear-cut column of discrete labelling across layers II–VI of the lateromedial field of the secondary visual cortex, rostral to the tracer injection site. The double asterisk marks an extended band of retrogradely labelled cells at the layer V/VI border of both the primary and the secondary visual cortex, rostral to the tracer injection site. Notice that labelled inverted neurons mainly lie in layers V–VI in the clear-cut column. Most labelled cells are inverted neurons in the extended band. (d) High-magnification image of labelled cells in the extended band of labelling depicted in (c). The distinctive morphologies of the spiny inverted neurons are apparent, not all of them being clearly ‘pyramidal’. Some of them have forked apical dendrites. Scale bars: (a,b) 100 µm; (c) 250 µm; (d) 30 µm.