Cofilin 2 is expressed in distinct classes of inhibitory neurons

96

Fig. 22: Analysis of Cofilin 2 protein levels at developmental steps during synaptogenesis in wt animals. Protein lysates of different brain regions of wt animals were prepared at P0, P7, P15, P21 and P90 and 15µg were loaded on a 15% SDS gel. The membrane was incubated with a specific antibody for Cofilin 2 (FHU-I). GAPDH served as a loading control. The obtained Western-blot signals for the Cofilin 2 expression were normalized to the obtained GAPDH signal for every probe with the Multi Gauge software. The average was calculated (n = 3). To compare the obtained Cofilin 2 level in different brain regions, the result for the P0 cortex was set as 100% and expression level of other regions were calculated based on the P0 cortex. The standard error was calculated.

In summary the highest expression of Cofilin 2 for all analyzed brain regions was dispayed at the onset of synaptogenesis around P7, except for the cortex and cerebellum. Interestingly, around this time point also the complete knockout of Cofilin 2 becames lethal.

4.1.4. Analysis of the Cofilin 2 expression in different

97

functional systems (van Brederode et al., 1991). Parvalbumin is localized to fast-spiking, non-adapting interneurons, while calbindin is present in regular-spiking interneurons (Markram et al., 2004). To analyze the expression of Cofilin 2 in inhibitory neurons immunofluorescence stainings with the Cofilin 2 specific antibody FHU-I and parvalbumin or calbindin on adult wt vibratome sections were prepared (figure 23 and 24).

An overlap in the expression of calbindin and Cofilin 2 was observed in different subtypes of inhibitory neurons. Thereby the Cofilin 2 staining in interneurons was localized to the cell soma, but was also observed in the nucleus. A first region that was analyzed for a colocalization between Cofilin 2 and inhibitory neurons was the cerebellum. Calbindin-positive cells in the cerebellum were exclusively found in the Purkinje cell layer, which only contains one type of inhibitory Purkinje cells. These Purkinje cells were also positive for Cofilin 2. Additionally a few Cofilin 2-positive cells were found in the molecular layer, which contains stellate and basket cells. A second region that was analyzed for the colocalization between calbindin and Cofilin 2 was the cortex. In the cortex calbindin positive cells in the layer II/III of the motor cortex were analyzed, which marks them as inhibitory smooth non-pyramidal neurons. Calbindin mainly marks double bouquet cells and Martinotti cells in the cortex. Thereby Martinotti cells are found in layer II – VI and are specialized in projecting their axons towards layer I, where they inhibit the tuft dendrites of pyramidal neurons.

Additionally their axons can also project horizontally in layer I to inhibit dendrites in neighboring columns and are thereby the only source for cross-columnar inhibition (DeFelipe 2002). These cells can also target multiple domains including proximal and perisomatic dendrites, as well as cell somata. Thereby Cofilin 2 could fulfill an important function in the remodeling of the actin cytoskeleton in presynaptic terminals to regulate synaptic vesicle release that controls firing patterns of pyramidal neurons in layer I. In contrast, double bouquet cells are only dendritic-targeting cells with a bitufted dendritic morphology that seems to be involved in the inhibition of basal dendrites (DeFelipe et al., 1990). Their axons branch frequently to form higher-order branches with densely studded boutons. These calbindin-positive cells were also positive for Cofilin 2, showing that Cofilin 2 was expressed in smooth non-pyramidal neurons. In these cells Cofilin 2 could fulfill a function in the actin remodeling during the dendritic arborization and axon branching process. But most of the cells in the cortex were exclusively positive for Cofilin 2, which leads to the suggestion that Cofilin 2 could be also localized to excitatory pyramidal cells, which represent the main class of neurons in the cortex. Another possibility would be excitatory spiny stellate cells, which are not positive for calbindin. In the third analyzed region, the striatum, Cofilin 2 colocalized with calbindin-positive cells that mark medium spiny neurons (Bennett and Bolam 1993). These medium spiny neurons are GABAergic and make up 90% of all cells in the striatum. Besides the localization of Cofilin 2 to the cell soma, a punctate staining of FHU-I could be also

98

examined in the striatum, which could result from afferent terminal endings, which innervate the striatum.

Fig.23: Cofilin 2 is expressed in Purkinje cells of the cerebellum, smooth non-pyramidal neurons in the cortex and in medium spiny neurons of the striatum. A Nissl staining of a sagittal mouse brain section. Analyzed regions were marked. B Vibratome sections of adult wt animals were stained with the antibodies FHU-I for Cofilin 2 and Calbindin, which marks inhibitory neurons. The cerebellum, cortex and striatum were analyzed for a possible colocalization between Cofilin 2- and Calbindin-positive cells. The FHU-I staining was detected via the secondary antibody Alexa488, and the Calbindin staining via Alexa594. Nuclei were stained with Draq5 and are shown in blue. Yellow arrows indicate cells which showed a colocalization between both antibodies, but not all FHU-I positive cells displayed also a staining for Calbindin. Images were obtained at 63x magnification.

Scale bar: 20µm

99

A second marker for a subset of inhibitory neurons is parvalbumin, which is also a calcium-binding protein that is localized to fast-spiking interneurons. The parvalbumin antibody displayed a punctate neuropil labeling and a strong staining of the cell soma.

A colocalization between parvalbumin and Cofilin 2 was analyzed in the cerebellum. In figure 23 Cofilin 2-positive cells were found in the molecular layer, which were not stained by calbindin. These cells can be marked by parvalbumin and are inhibitory stellate and basket cells (figure 24). These cells in the molecular layer form GABAergic synapses onto dendritic branches of Purkinje cells. Therefore a colocalization between parvalbumin and FHU-I could be examined in these stellate and basket cells (figure 24). Additionally parvalbumin was also localized to Purkinje cells, which displayed again a colocalization with the Cofilin 2 antibody.

Parvalbumin is also expressed in principal sensory and motor relay nuclei in the thalamus, which project back to the cortex and form the sensory input that is used as a basis for perception (Jones and Pons 1998). In these cells a Cofilin 2 expression was also examined.

Additionally a few more cells were positive for Cofilin 2, which were not stained by parvalbumin. These could be relay cells which were only positive for calbindin (data not shown) and project to layer I of the somatosensory cortex (Rausell et al., 1992). These calbindin positive cells form a basis for the engagement of multiple aspects of sensory experience into a single framework of consciousness. In the thalamus nuclei could be distinguished by different patterns of parvalbumin- and calbindin-positive cells, and reflect that thalamic cells belong to different functional systems that also project differentially into the cortex (Magnusson et al., 1996). Cofilin 2 was expressed in both analyzed thalamic systems, since principal sensory and motor relay cells (marked by parvalbumin) and relay cells stained by calbindin were positive for Cofilin 2.

In the striatum we already confirmed the expression of Cofilin 2 in medium spiny neurons. A second cell type in the striatum are fast-spiking GABAergic interneurons, which participate in feed-forward inhibition of principal neurons and are positive for parvalbumin. In these cells no colocalization between FHU-I and parvalbumin was observed, excluding the expression of Cofilin 2 in fast-spiking interneurons (figure 24).

In summary Cofilin 2 was localized to a subset of inhibitory neurons in different brain regions, but no expression of Cofilin 2 was detected in fast-spiking inhibitory neurons.

100

Fig.24: Cofilin 2 is expressed in stellate and basket cells of the cerebellum, principal sensory relay nuclei in the thalamus, but not in fast spiking interneurons of the striatum. A Nissl staining of a sagittal mouse brain section. Analyzed regions were marked. B Vibratome sections of adult wt animals were stained with the antibodies FHU-I for Cofilin 2 and parvalbumin, which marks inhibitory neurons. The FHU-I staining is displayed via the secondary antibody Alexa488, and the parvalbumin staining via Alexa594. Nuclei were stained with Draq5 (blue). The cerebellum, thalamus and striatum were analyzed for a possible colocalization between Cofilin 2- and parvalbumin-positive cells. Images were obtained at 63x magnification. Scale bar: 20µm