[page 42↓]

3.  Results

3.1. Preparation of monospecific and affinity-purified antibodies

3.1.1. Comparison of the amino acid sequences

Functional Kir2 channels are formed by tetrameric association of subunits. Each subunit contains two membrane-spanning regions M1 and M2. They flank a highly conserved pore-forming loop called H5. In order to produce specific antibodies against individual members of the Kir2 family, unique amino acid segments are needed. Comparison of the complete sequences of the Kir2 subfamily (Fig. 7) shows strong homology between the members in the transmembrane domains, the pore region and the adjoining cytoplasmic amino acids (shaded red). The most significant differences exist at the carboxyl terminal regions, making them the best target for antibodies. These less conserved sequences (42-68 amino acid residues, shaded gray in Fig. 7) were selected as fusion proteins for subsequent cloning and then for raising of polyclonal antibodies in rabbits.

The selected sequences were also searched for homologies with other proteins using the protein databases Swiss-Prot, NCBI-BLAST and TrEMBL. The Kir2.3 sequence contains two known domains, namely the Poly-Glu domain in amino acid position 383-390 and the Poly-Ala domain in position 391-399. There were no such domains in the sequences of the Kir2.1, Kir2.2 and Kir2.4 protein, respectively. The Poly-Ala-Poly-Glu domain or closely related domains were found in human ALMS1 protein, microtubule-associated protein EB1 (Arabidopsis thaliana), human PEG3 protein, different zinc finger proteins, mouse growth factor receptor bound protein 2-associated protein 3, NBS-LRR-like protein (Oryza sativa), human KIAA1855 protein and human mixed lineage kinase.

If there was a cross reaction to these proteins, we should detect a signal in non-transfected COS7 mammalian cells, as some of these proteins, the zinc finger domains in particular, are present in virtually all mammalian cells. However, the anti-Kir2.3 antibody only stained cells that were transfected with the Kir2.3 DNA while control cells remained unstained (not shown). This finding is confirmed by Western blots. Instead of multiple bands only a single band can be detected in the expected range of the Kir2.3 channel protein (see Fig. 14). [page 43↓]Thus, the affinity purified anti-Kir2.3 antibody does not detect certain domains in other proteins.

Fig. 7: Amino acid sequences of Kir2 subfamily members. The primary structures of all rat Kir2 channel subunits demonstrate that sequence conservation is lowest in the carboxyterminal area. Sequences shared by all four subfamily members are labeled in red. The carboxy terminal sequences uses for antibody production are depicted in gray. M1, M2 transmembrane regions; P5 pore-forming region (adapted from Töpert 1998)


[page 44↓]

3.1.2.  Recombinant fusion proteins

After PCR amplification, the carboxyl terminal regions were cloned into the procaryotic expression vectors pQE40 and pGEX-4T-1 applying restriction enzymes. After plasmid transformation, positive clones were selected and protein expression in E. coli bacteria was performed. Fig. 8 displays the purified fusion proteins that were used for rabbit injections in two animals with different doses.

Fig. 8: SDS-PAGE of purified fusion proteins Kir2.1-Kir2.4

3.1.3. Removal of IgM antibodies by gel-filtration

Antibodies of the IgM class cannot be detected by applying further secondary anti-rabbit antibodies. However, they may reversibly block epitopes and prevent the binding of IgG antibodies, thereby decreasing the specific signal. Therefore these immunoglobulins need to be removed. Rabbit sera with the highest titer of specific Kir2 antibodies were pooled. Separation of IgG and IgM was performed by passage of the Superdex-column making use of the wide difference in molecular weight. The example of the Kir2.2 antibodies (Fig. 9) demonstrates the procedure of fraction selection: The protein line measured photometrically at 280nm (green line) displays the large IgM in the first peak (fractions 20-24). An ELISA measurement of the immunoreactivity to the Kir2.2 antigen (red line) leads [page 45↓]to the fractions with highest IgG concentration. Fractions 24-36 were pooled and used for further purification of specific antibodies and as a control of cross reactivities among the Kir family. An analogous procedure was used with remaining Kir2 subfamily members.

Fig. 9: Elution profile after passage through a superdex column. The green line represents the protein concentration and the red line the immunoreactivity to the corresponding Kir2 protein. Fractions selected on the basis of high antibody titer (24-36) are marked.

3.1.4. Affinity purification

The remaining solution contains antibodies specific to the Kir2 channel protein, but of course also to the rest of the fusion protein. Therefore absorption of these unintended antibodies was achieved by applying pQE40 or pGEX protein as the next step of purification. The optimal amount of protein was determined by testing the decreased cross reactivity depending on added protein concentration (Fig. 10A, arrow). Moreover, to get the anti-Kir2 antibodies, solely affinity purification was performed making use of a second expression system (pet32b); hence a different protein pattern surrounding the antigenic sequence. Nitrocellulose membranes were coated with this new fusion protein. Hence only antibodies to the Kir2 protein could bind and subsequently become washed out (compare [page 46↓]methods).
Fig. 10: Affinity purification. A) Initial application of pQE40 protein only to absorb cross reactivity to the plasmid part of the expressed antigen. B) Varying conditions to find protein concentration with best antibody quantity.

Previously, optimal conditions for affinity purification were tested to find an effective ratio between applied antigen amount and purified antibody concentration. Exemplary in the case of Kir2.2 (Fig. 10B) the highest amount of antibodies was collected using 5 µg protein/ cm2 (arrowhead). Nevertheless only about 25% of starting activity was detectable, probably because of a high antibody affinity for the protein tethered to the membrane. Even more radical elution conditions (decreased pH) could not increase the quantity of purified antibodies. The sera of the remaining Kir2 proteins were processed using the same method and parameters.

3.2. Specificity of purified antibodies

3.2.1. Cross reactivity

Prior to affinity purification, immunoreactivity is present to the common part of the fusion protein. Afterwards all four anti-Kir2 antibodies failed to show any cross reactivity to the [page 47↓]other subfamily members. Additionally, cross-reactivity concerning the related Kir3 subfamily was excluded (Fig. 11).

Fig. 11: No cross reactivity to other Kir subunits. Microtiter plates were coated with different proteins of Kir channels. Antibodies of the Kir2 subfamily exclusively detect their corresponding antigen and display no cross reactivity to other subunits. The horizontal line indicates extinction of BSA controls.

3.2.2. Competitive ELISA

Antibody quality was evaluated at several further levels following affinity purification. Specificity for the corresponding primary sequences is shown best in the competitive ELISA assay (Fig. 12). It shows on the one hand that a specific polyclonal antibody cannot be blocked by proteins of other Kir2 subfamily members, on the other hand that only the distinctive protein is able to reduce the immunoreactivity depending on the concentration used. This knowledge of specific recognition of native proteins is essential for antibody application to rat brain tissue sections.


[page 48↓]

Fig. 12: ELISA assays of all members of the Kir2 subfamily. Microtiter plates were coated with the respective recombinant protein. Preincubation of antibodies with the cognate antigen only decreases the immunoreactivity in a concentration-dependent manner. Preincubation with other antigens did not show any effect.

3.2.3. Western blot of fusion proteins

A detection of denaturated fusion proteins was verified in Western blots supporting the results from the competitive ELISA. Reaction to all members of the Kir2 subfamily was examined. Again each antibody recognizes its corresponding antigen only (Fig. 13).


[page 49↓]

Fig. 13: Specificity of anti-Kir2 antibodies. In Western blots Kir2 antibodies recognize only their corresponding fusion protein and none of the other Kir2 subfamily members.

3.2.4. Western blot of rat brain homogenates

Competitive ELISA testing does not exclude cross reactivities with other cellular proteins. Therefore, the next step of specificity can be investigated by performing Western blot analysis of rat brain homogenates (Fig. 14). Only a single band in brain proteins suggests detection of the correct channel and the band is supposed to be located in the range of the expected molecular weight of the channel protein. All of the purified antibodies detect a specific band (left lane) that can be blocked by preincubation of the antibody with their fusion protein (right lane). In the case of Kir2.1, Kir2.2 and Kir2.3, the recognized protein displays the expected molecular weight as predicted from amino acid sequences.


[page 50↓]

Unfortunately, convincing Western blots from brain homogenates were not obtained with the anti-Kir2.4 antibodies that detect a single band with a molecular weight much higher than expected (see next paragraph).

Fig. 14: Specificities of the anti-Kir2 antibodies as judged from Western blots. Homogenates from total rat brain membranes (10 µg/lane) were separated on 10% SDS-PAGE. All antibodies recognized single bands of the expected molecular weights (left lanes; with exception of Kir2.4, see next chapter) which were largely abolished subsequent to preincubation block with the cognate recombinant proteins (right lanes).

3.2.5. Specificity of anti-Kir2.4 antibodies

The surprising difference between the sizes of predicted protein versus obtained band with anti-Kir2.4 antibodies in western blot of rat brain homogenates (Fig. 14) required supplementary examination.

In order to verify the specificity of the anti-Kir2.4 antibodies, COS-7 cells were transiently transfected with Kir2.4-DNA (Fig. 15A). In contrast to untreated controls (Fig. 15B), incubation of transfected cells with anti-Kir2.4 antibodies visualized a remarkable fraction of positive cell bodies. Consequently, these antibodies recognize the correct antigen. To confirm the finding, a Western blot of homogenate of transfected cells was performed (Fig. 15C): only COS cells expressing the Kir2.4 channel display a protein band of predicted size (left lane), whereas no band is seen when blotting non-transfected cells (right lane). [page 51↓]Whether the fainter second band represents a phosphorylated form or a minor breakdown product remains unclear at present.

So far, there are different possibilities causing the single band in the Western blot of exact double the size of the potassium channel monomer. On the one hand the antibody might cross-react with a yet unknown brain specific protein. On the other hand the band may represent a Kir2.4 dimer that did not further dissociate during SDS-treatment before the Western blot (see also discussion in chapter 4.1.). Further examinations are required to finally rule out cross-reactivity.

Fig. 15A,B: Specificity of anti-Kir2.4 antibodies, transfection experiments. The specificity of the Kir2.4 antibodies was further verified by the immunofluorescence obtained with Kir2.4 transfected COS-7 cells (A). Non-transfected cells were negative (B).

Since the Kir2.4 channel was reported to be expressed in the heart, an additional Western blot analysis of heart homogenate was done. It shows a protein band within the correct range (Fig. 15D), thus the anti-Kir2.4 antibodies recognize the correct protein size again. It is of remarkable interest that the Kir2.4 channel can be found only in neuronal structures of the heart1.


[page 52↓]

Fig. 15C,D: Specificity of anti-Kir2.4 antibodies. Western blots from transfected cells (C) displayed a double band (left lane), most probably due to the presence of phosphorylated species, within the expected range of molecular weights. Untransfected cells yielded blank blots (C, right lane). Western blots of heart homogenate (D) also show a protein band within the correct range.

3.2.6. Antibody specificity in brain sections

Immunocytochemical specificity was demonstrated using the olfactory bulb as an example (Fig. 16). The anti-Kir2 antibodies were applied to 20 µm rat brain sections. Each antibody showed a characteristic and individual distribution pattern. In all cases staining was abolished subsequent to preincubation with the cognate recombinant protein (not shown here, but see Fig. 23C). Chosen concentrations were used in all of the light microscopic screening experiments.


[page 53↓]

Fig. 16: Immunocytochemical labeling of the rat olfactory bulb confirms the specificity of the anti-Kir2 antibodies. For detailed analysis of the immunocytochemical specificities of the anti-Kir2 antibodies an area (boxed in A) containing the outer layers of the olfactory bulb was selected. Individual layers are evident at higher magnification in the section stained with cresyl violet (B). Immunoreactivities of individual members of the Kir2 subfamily (C-F) display specific and individual distribution patterns. Gl, glomerular layer; EPl, external plexiform layer; Mi, mitral cell layer; IPl, internal plexiform layer; Gr, granule cell layer.

The immunocytochemical localizations of the Kir channel proteins were in good agreement with in situ hybridization data2. Thus, Kir2.1 immunostaining was most prominent in the [page 54↓]glomerular layer (Fig. 16C), where also Kir2.1 mRNA is intensively expressed3. Again in agreement with mRNA findings, Kir2.2 immunoreactivity was most prominent in the glomerular layer and only weakly detectable in the mitral cell layer. Contrasting the other subfamily members, Kir2.3 channel proteins were predominantly expressed in the external plexiform layer of olfactory bulb and were virtually absent from the periglomerular cells (Fig. 16E). This is in perfect agreement with recent data from Kurachi’s group4. Kir2.4 staining was found in most neurons, especially in the large cell bodies of mitral and tufted cells. Interestingly, in the granule cell layer, prominent immunostaining was displayed only by a small subset of granular cells (Fig. 16F, arrows).

3.3. Distribution of Kir2 channels in the rat brain

The expression of Kir2 mRNAs in the rat brain is known from in situ hybridization (ISH) experiments5. The corresponding proteins, however, may not necessarily be found in the same area, but may instead be sorted to the axons or their distant terminals. ISH data, therefore, do not necessarily display the subcellular and regional distribution of the channel protein and require complementary information from immunocytochemical experiments. Consequently, in a first step our experimental work focused on the general distribution of Kir2 channel proteins in different brain regions (Fig. 17-I – 17-IX).

3.3.1. Olfactory system

All four Kir2 subunits were expressed in the olfactory bulb (see detailed findings in chapter 3.2.6. and Fig. 16). Although all channels were expressed in the piriform cortex, the olfactory tubercle (Fig. 17-I) was labeled prominently by Kir2.1 and Kir2.3 antibodies and to a lesser extent by the other channel proteins. This finding is in excellent agreement with former data from in situ hybridization experiments6.


[page 55↓]

Fig. 17-I: Olfactory tubercle. For details see text. cc, corpus callosum; IG, induseum griseum; CPu, caudate putamen; AcbC, core part of the accumbens nucleus, AcbSh, shell part of the accumbens nucleus; Tu, olfactory tubercle; Pir, piriform cortex.


[page 56↓]

3.3.2.  Hippocampus

The most prominent Kir2 channel expression was detected in the dentate gyrus (Fig. 17-II), although the Kir2.4 subunit was labeled to a lesser extent. Nevertheless, staining intensities varied also among the Kir2.1-2.3 members. Kir2.1 was present in the granule cell layer with particularly elevated levels, whereas the specific signal in all other hippocampal layers was very low, also including the neurons of the Ammon’s horn. In contrast, the Kir2.2 subunit was found to be markedly elevated in the molecular layer of the dentate gyrus. Although all four Kir2 subunits were virtually absent from CA3 neurons, a prominent labeling of CA1 and CA2 neurons was detectable by Kir2.2 and Kir2.3 antibodies. Again the Kir2 channel distribution within the hippocampus is in good conformity with former in situ hybridization experiments7.

3.3.3. Neocortex

Kir2.1-2.4 channels were expressed in all cortical areas (Fig. 17-III,B-E) and the antibody signal could be seen in most of the cortical cells, suggesting a possible co-expression of the different Kir2 channels. The labeling pattern reflects the laminar structure of the rat neocortex when compared with a Klüver-Barrera-stained section (Fig. 17-III,A). The strongest immunoreactivity was shown by Kir2.1 and Kir2.3 with a focal point on layer II and III neurons. Kir2.2 protein was present at moderate levels in all layers, with particularly elevated levels in layer V pyramidal cells. The Kir2.4 subunit was expressed in virtually all cortical neurons, but with only weak immunoreactivity. This latter finding partly differs from the distribution pattern described by Töpert8, who reported the Kir2.4 subunit to be absent from the rat cortex (see also discussion).


[page 57↓]

Fig. 17-II: Hippocampus. Areas C-F are boxed ‘a’ in A. For details see text. CA1-CA3, cornu ammonis 1-3; DG, dentate gyrus; fi, fimbriae; MHb, medial habenula; LHb lateral habenula.


[page 58↓]

Fig. 17-III: Cortex (A-E), habenula (F-J). Areas A-E are boxed ‘b’ and F-J corresponds to ‘c’ in Fig. 17-IIA. For details see text.


[page 59↓]

Fig. 17-IV: Nucleus accumbens. For details see text. cc, corpus callosum; CPu, caudate putamen; AcbC, core part of the accumbens nucleus, AcbSh, shell part of the accumbens nucleus; Tu, olfactory tubercle; Pir, piriform cortex; aca, anterior commissure.

3.3.4. Basal ganglia and amygdala

The caudate putamen and nucleus accumbens (Fig. 17-IV) were reported to be among the most prominently labeled brain structures when probed with Kir2.1 and Kir2.3 mRNAs9, which is in good agreement with our antibody studies (the differential channel distribution in the striatum is summarized in chapter 3.4.1.).


[page 60↓]

Fig. 17-V: Thalamus. For details see text. Areas G and H are boxed in A. CPu, caudate putamen; GP, globus pallidus; ic, internal capsule; Rt, reticular nucleus; sm, stria medullaris; Pir, piriform cortex; LOT, lateral olfactory tract nucleus; SO, supraoptic nucleus; ox, optic chiasma.


[page 61↓]

Kir2.4 was only weakly detectable in the nucleus accumbens, whereas all the other Kir2 channel subunits were abundantly expressed. Kir2.1 and Kir2.2 proteins were present in all accumbal parts at moderate levels, whereas a more differential signal was observed for Kir2.3, with particularly elevated levels in the shell part of the nucleus accumbens (Fig. 17-IV,E) and a patchy distribution resembling the expression pattern within the striatum (compare chapter 3.4.1.). Although Kir2.2-2.4 proteins were absent in amygdala nuclei and lateral olfactory tract nucleus, significant levels of Kir2.1 were found in the amygdala and strong Kir2.1 expression in the lateral olfactory tract nucleus (Fig. 17-V,C).

3.3.5. Thalamus

In the thalamus, all four Kir2 subunits displayed a differential distribution pattern. The Kir2.4 subunit was virtually absent in all thalamic nuclei. The Kir2.3 protein was also not detectable in most of the nuclei (anterior, lateral, paraventricular, posterior, medio-, laterodorsal, medial geniculate etc.), but it was expressed in the reticular thalamic nucleus (Fig. 17-V,E), which supports former mRNA studies10. The distribution of the Kir2.1 channel subunit, however, is in contrast to these in situ hybridization experiments, where the Kir2.1 protein is reported to be absent from all thalamic nuclei. The present study reveals a strong signal in the anterodorsal nucleus (Fig. 17-V,C) and still moderate levels for example in anteroventral, reticular and posterior thalamic nuclei. As previously described, the Kir2.2 subunit was abundant in many thalamic nuclei.

3.3.6. Hypothalamus and habenula

In conformity with earlier mRNA labeling11, the Kir2.2 channel subunit was expressed most prominently in hypothalamic nuclei with the highest signal being observed in anterior area, preoptic nucleus and supraoptic nucleus (Fig. 17-V,G). The Kir2.3 protein was absent in the hypothalamus and only weak expression was observed with Kir2.4 and Kir2.1 antibodies. The exception was the Kir2.4 immunoreactivity in the supraoptic nucleus, where particularly elevated levels were found. Only one subunit, Kir2.2, was present at [page 62↓]extremely high levels in the medial habenula, but not in the lateral habenula (Fig. 17-III,H). Kir2.1 protein was found throughout the habenula, but with significantly lower levels when compared with Kir2.2. In contrast, the Kir2.4 channel subunit was absent and the Kir2.3 protein was only weakly expressed in the medial habenula.

Fig. 17-VI: Substantia nigra. For details see text. MG, medial geniculate; CG, central gray; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; cp, cerebral peduncle; IP, interpeduncular nucleus.


[page 63↓]

3.3.7.  Substantia nigra, ventral tegmental area (VTA), superior colliculus

All four Kir2 channel subunits were expressed by substantia nigra cells in a differential pattern (Fig. 17-VI). The Kir2.1 and Kir2.4 subunit was observed only in the SN pars compacta, whereas the Kir2.2 and Kir2.3 proteins were expressed in both parts auf SN with markedly elevated levels in the pars reticulate, Kir2.2 and Kir2.3 displaying a similar distribution pattern, indicating co-expression of these two subunits. Only one channel protein, Kir2.1, was present at low levels in the ventral tegmental area.

The superior colliculus (optic tectum) was among the most prominently labeled brain structures when labeled with Kir2.1 antibodies (Fig. 17-VII,C) and displayed also elevated signals for the Kir2.2 subunit protein. In contrast, the Kir2.3 subunit was only weakly expressed in this major target for retinal ganglion cells and the Kir2.4 channel protein was absent. Expression in the central gray was generally weak; however, few cells were expressing elevated levels of Kir2.2 protein. Otherwise, Kir2.2 and in this isolated case Kir2.4 channels were extensively expressed in the oculomotor nucleus (Fig. 17-VII,H/J), which is in line with with mRNA studies12.

3.3.8. Cerebellum and spinal medulla

The Kir2 channel proteins present remarkable differences in the expression pattern throughout the cerebellum (Fig. 17-VIII). The Kir2.2 subunit expression was the strongest in the cerebellar cortex with restriction to the granule cell layer, thereby supporting the former data from in situ hybridization13. Contrasting this previously reported mRNA distribution, the Kir2.3 channel protein was entensively expressed by the large neurons in the deep cerebellar nuclei and by Purkinje cells, which is, however, in agreement with data from Falk’s group14. The molecular layer contained low levels of Kir2.1 and Kir2.4 proteins. Both channels were also weakly expressed in the deep nuclei of the cerebellum. All Kir2 subunits were present in the motoneurons of the spinal cord with particularly [page 64↓]elevated levels of the Kir2.2 channel protein.
Fig. 17-VII: Mesencephalon. Adjacent sections of the oculomotor nucleus (G-J) are from the region boxed in A. For details see text. MG, medial geniculate; SuC, superior colliculus; CG, central gray; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area; cp, cerebral peduncle; IP, interpeduncular nucleus; 3, oculomotor nucleus.


[page 65↓]

Fig. 17-VIII: Cerebellum. Adjacent sections from the cerebellar cortex (C-F) and deep nuclei (G-J). For details see text. Med, medial cerebellar nucleus; Int, interposed cerebellar nucleus; Lat, lateral cerebellar nucleus.


[page 66↓]

Fig. 17-IX: Medulla spinalis. For details see text. cu, cuneate fasciculus; lfu, lateral funiculus; vfu, ventral funiculus; 2-9, spinal cord layers.

The previously reported preference of hindbrain structures to express the Kir2.2 subunit15 can be supported by the finding of Kir2.2 protein throughout the gray matter of the spinal cord with extremely high levels in the substantia gelatinosa.


[page 67 - 68↓]

Table 5: Distribution of individual Kir2 subunits in different brain regions

Brain region

Kir2.1

Kir2.2

Kir2.3

Kir2.4

Telencephalon

    

Olfactory bulb

    
 

Glomerular layer

++++

+

0

+

 

External plexiform layer

+

0

++++

0

 

Mitral cell layer

+

+

+

++

 

Granule cell layer

++

0

+

+

Olfactory tubercle

++

+

+++

0

Islands of Calleja

+

0

0

0

Piriform cortex

++

+

++

+

Neocortex

    
 

Layer I

0

+

+

0

 

Layer II

+++

++

+++

++

 

Layer III

+++

++

+++

+

 

Layer IV

++

+

++

++

 

Layer V

++

+++

++

++

 

Layer VI

++

+

+

+

Hippocampus

    
 

Dentate gyrus

    
  

Molecular layer

+

+++

+

0

  

Granule cell layer, Lamina sup.

+++

++

++

+

  

Granule cell layer, Lamina inf.

0

+

+

0

  

Polymorphic layer

    
 

CA1-CA2

    
  

Oriens layer

0

+

++

+

  

Pyramidal cell layer

+

++

++

+

  

Radiate layer

0

+

++

0

  

Lacunosum moleculare layer

+

++

+

0

 

CA3

    
  

Oriens layer

0

+

+

+

  

Pyramidal cell layer

+

+

+

+

  

Radiate layer

0

+

+

+

Basal forebrain

    
 

Substantia innominata

0

+

0

+

 

Basal nucleus of Meynert

+

++

++

+

Basal ganglia

    
 

Caudate putamen (Striatum)

++

+++

+++

+

 

Nucleus accumbens

++

++

++

+

 

Globus pallidus

+

++

++

+

 

Claustrum

0

+

0

+

 

Endopiriform nucleus

+

+

+

+

Lateral olfactory tract nucleus

+++

0

+

+

Amygdala

    
 

Cortical nuclei

+

0

0

0

 

Medial nucleus

+

0

0

0

 

Basomedial nucleus

+

0

0

0

     

Diencephalon

    

Thalamus

    
 

Anterodorsal nucleus

+++

0

0

0

 

Anteroventral nucleus

++

0

0

0

 

Anteromedial nucleus

+

0

0

0

 

Laterodorsal nucleus

+

+

0

0

 

Mediodorsal nucleus

+

0

0

0

 

Centrolateral nucleus

0

++

0

+

 

Paraventricular nucleus

+

++

0

+

 

Reuniens nucleus

+

+

0

0

Reticular nucleus

++

++

++

+

 

Posterior nucleus

++

+

0

0

 

Medial geniculate nucleus

+

+

0

0

 

Paratenial nucleus

+

++

0

+

Hypothalamus

    
 

Anterior area

+

++

0

+

 

Ventrolateral nucleus

0

+

0

+

 

Lateral area

+

+

0

0

 

Preoptic nucleus

+

++

0

+

 

Periventricular nucleus

+

+

0

0

 

Paraventricular nucleus

+

+

0

+

 

Supraoptic nucleus

+

++

0

++

Epithalamus

    
 

Medial habenula

++

++++

+

0

 

Lateral habenula

+

+

0

0

     

Mesencephalon

    

Red nucleus

0

+

+

+

Substantia nigra

    
 

Pars compacta

+

++

+

+

 

Pars reticulata

0

+++

++

0

Ventral tegmental area

+

0

0

0

Interpeduncular nucleus

++

+++

++

0

Superior colliculus

++++

++

+

0

Central gray

+

++

0

+

Edinger-Westphal nucleus

+

+

0

+

Oculomotor nucleus (III)

+

++

0

+++

     

Metencephalon

    

Cerebellum

    
 

Deep nuclei

+

0

+++

+

 

Molecular layer

+

0

0

+

 

Granule cell layer

0

+++

0

+

 

Purkinje cells

0

0

++

0

     

Myelencephalon

    

Spinal medulla

    
 

Substantia gelatinosa

+

++++

+

0

 

Motoneurons

++

+++

+

+

3.4. Distribution of Kir2 channels in the striatum

The striatum plays a critical role in movement selection, motivated behavior and neural transmission via basal ganglia circuitry. To evaluate the possible involvement of Kir2 channels in the fine tuning of these pathways, it is important to know their exact distribution within the striatum. A distinct localization in either the patch compartment or the cholinergic interneuron would suggest that selective channel activation or inhibition could specifically influence the outcome of a movement and, perhaps, open new strategies for novel therapeutic regimes.


[page 69↓]

Table 6: Striatal Kir2 channel expression, results from in situ hybridization experiments

channel subunit

author

remarks

Kir2.1

Karschin, C. et al. 1996

abundant (Acb, CPu)

Kir2.2

Karschin, C. et al. 1996

moderate (Acb, CPu), large cells prominent

Kir2.3

Karschin, C. et al. 1996

abundant (Acb, CPu)

 

Falk, T. et al. 1995

only few (VP, CPu)

 

Bredt, D.S. et al. 1995

high (striatum)

Kir2.4

Töpert, C. et al. 1998

no expression

Abbreviations: Acb accumbens nucleus, CPu caudate putamen, VP ventral pallidum

3.4.1. Kir2 subunits are differentially distributed in the rat striatum

The expression of Kir2 mRNAs in the rat striatum is known from in situ hybridization experiments (table 5). Immunocytochemical experiments were performed to get more detailed information about the subcellular and regional Kir2 channel protein distribution.

Rat brain sections were stained with antibodies to all four Kir2 subunits using the routine conditions described above. Although distribution in some regions of the rat brain is very divergent, in the striatum there are remarkable similarities: Survey micrographs (Fig. 18) suggest that all four Kir2 channel proteins are expressed by medium-sized spiny neurons. Nevertheless, staining intensities varied among the members of the potassium channel subfamily. The strong Kir2.2 staining of the neuropil in the caudatoputamen (CPu) obscured the visualization of neuronal cell bodies (Fig. 18D). Indeed, many spiny projection neurons were moderately labeled with no obvious preference for a subset of cells. Interestingly, neurons with prominent Kir2.2-immunoreactivity (Fig. 18D, arrowheads) were grouped in the pallidum (GP). The same was true for the Kir2.3 channel (Fig. 18E). Not all neurons of the pallidum are stained with the same intensity, suggesting functional differences. Whether the strongly stained neurons represent a cell type similar to the newly described type II neurons in the internal pallidum16 is unclear at present.


[page 70↓]

Fig. 18: Regional and cellular distribution of Kir2 subunits in the rat striatum. A series of six adjacent sections was used to analyze the immunocytochemical patterns of the individual members of the Kir2 family. Coronal sections were obtained shortly caudal to the optic chiasm and stained according to Klüver-Barrera for morphological examination (A,B). An area (boxed in A) containing parts of the caudate-putamen and the external pallidum was selected for further investigation. Distinct patterns of immunostaining were obtained for the individual Kir2 subunits (C-F). Note that Kir2.2-positive neurons are clustered in the pallidum (D, arrowheads) and that large neurons in the striatum display prominent staining (F, arrows) with the anti-Kir2.4 antibody. CPu, caudate-putamen; GP, globus pallidum externum; Pir, piriform cortex; sm, stria medullaris thalami.

The Kir2.1 subunit is apparently restricted to the somatodendritic compartment of many, if not all, spiny projection neurons leaving the neuropil unstained (Fig. 18C). Neurons of the pallidum also displayed homogeneous immunoreactivities. The Kir2.3 distribution pattern [page 71↓]of the caudatoputamen resembles that of the Kir2.1 subunit. Many projection neurons are labeled. In contrast to the other subunits, however, Kir2.3 immunoreactivity displayed stronger and weaker stained areas (see below). Finally, the Kir2.4 channel subunit showed a unique staining pattern with considerably less immunoreactivity as compared to the other Kir2 proteins (Fig. 18F). Probably all striatal and pallidal neurons could be detected with the anti-Kir2.4 antibodies. Some large neurons of the caudatoputamen, however, displayed especially strong Kir2.4 immunoreactivity (arrows).

3.4.2. Anti-Kir2.2 antibodies stain striatal fiber bundles

Aside from somatodendritic labeling of projection neurons, Kir2.2 immunoreactivity also produced a dense signal in the neuropil (Fig. 18). Under routine conditions (see methods) brain sections incubated with anti-Kir2.2 antibodies did not allow visual identification of stained fibers. However, a different image appeared when immunocytochemical conditions were optimized to focus on this special problem (Fig. 19). The lower concentration of anti-Kir2.2 antibodies hardly stained spiny projection neurons, but showed an extensive labeling of striatal fiber bundles, which was absent after preincubation with the Kir2.2 protein. This observation was consistent throughout rostral and caudal areas of the striatum and also in different animals. As an example, coronary sections of the striatum at the level of the anterior commissure (Fig. 19A, B) are depicted.

The sorting of the Kir2.2 protein to the axon compartment is supported by data obtained at the electron microscopic level. Gold dots represent Kir2.2 channel protein. They are highly concentrated within the fiber bundle (Fig. 19C), but only sparse in the surrounding neuropil.


[page 72↓]

Fig. 19: Anti-Kir2.2 antibodies stain striatal fiber bundles. Using carefully selected conditions (for details see text) Kir2.2 immunoreactivity displayed a prominent labeling of fiber bundles within the striatum (A,B). At the electron microscopic level (C) the gold dots representing Kir2.2 channel distribution are found highly concentrated within the fiber bundles, suggesting the Kir2.2 subunit being sorted to the axonal membrane. Gold dots are sparse in the surrounding neuropil.


[page 73↓]

3.4.3.  Kir2.3 channels are predominantly localized in the matrix compartment

The detailed analysis of the Kir2.3 protein distribution again required carefully selected antibody concentrations. These optimized conditions demonstrate that from the Kir2 family proteins only the Kir2.3 subunit is distributed inhomogeneously. Regions of low antibody binding are intermingled with areas of stronger immunoreactivity (Fig. 20A). This heterogeneity, which may be related to the compartmental organization, was detectable in all rats and extended throughout the whole striatum along the rostro-caudal axis.

Fig. 20: Kir2.3 channel subunit protein is inhomogeneously distributed throughout the striatum. Complete immunostained sections (A) of the rat forebrain, unlike sections with antigen competition (B), show Kir2.3 immunoreactivity in the striatum. Using selected conditions, an inhomogeneous distribution of the Kir2.3 subunit in the striatum becomes obvious (C). The pattern of reduced Kir2.3 staining seems similar to that of the striatal patch compartment as visualized via the immunocytochemical localization of µ-opiate receptors (D).

Now one could argue that the patchy distribution of Kir2.3 immunoreactivity might be caused by the existence of cell-dense and cell-poor regions. To rule out this possibility the Kir2.3 localization was compared to an adjacent Klüver-Barrera stained section (Fig. 21). [page 74↓]The medium spiny neurons are rather homogeneously distributed between the prominent fiber bundles, a typical feature of the striatum. In regions of strong Kir2.3 reaction (Fig. 21A and B, arrowheads) and in poorly stained areas (arrows), cell density is similar. Consequently, the patchy distribution of Kir2.3 channel subunits cannot result from high or low cell densities. In contrast, it seemed to be roughly congruent with the distribution of µ-opiate receptors (Fig. 20D), the original17 and well recognized marker of the striatal patch compartment.

Fig. 21: Striatal cell density. Adjacent sections stained according to Klüver-Barrera (A) and for the Kir2.3 subunit (B) demonstrate that the increased Kir2.3 immunoreactivity is not simply due to an increased density of nerve cells at this location (compare areas between the three arrowheads or between the four arrows). Note the identical localization of capillaries in (A) and (B).

The identity of the Kir2.3-weak areas with the patch compartment became obvious when both distribution patterns were compared in adjacent sections (Fig. 22). The upper images (Figs. 22A, C) display the distribution of patch compartments using immunocytochemical visualization of µ-opiate receptor distribution. The same areas in an adjacent section are shown in the lower panel photographs (Figs. 22B, D) stained with anti-Kir2.3 antibodies. Striatal fiber bundles are hatched to make sure that the patchy Kir2.3 reactivity is not confused with unstained fibers.

In an overlay of striatal patches with the Kir2.3 image (Fig. 22B, D) the low Kir2.3 immunoreactivity inside the patch compartment is verified. The lines surrounding the patches nicely correspond to the borders between areas of different Kir2.3 staining [page 75↓]intensities. Thus, there is a clear correlation between areas with increased Kir2.3 immunoreactivity and the striatal matrix compartment.

Fig. 22: Correspondence between the striatal patch compartments and areas with reduced Kir2.3 immunoreactivity. For direct correlation, areas of reduced Kir2.3 immunoreactivity (B,D) and the striatal patch compartment as visualized by µ-opiate receptor staining (A,C) are compared in adjacent sections. In (B,D) areas occupied by fiber bundles are hatched to avoid confusion. The patch compartment (as seen in A and C) is outlined in black to facilitate comparison.

3.4.4. Kir2.4 channel subunits are localized at cholinergic interneurons

In addition to the striatal patch compartment, cholinergic interneurons represent a major decision making component within the basal ganglia circuitry. Localization of one of the Kir2 channel subfamily members in these giant interneurons would imply their importance in the regulation of basal ganglia circuitry.

A survey micrograph (Fig. 23A) shows large Kir2.4-positive neurons (arrows) dominating the minor immunoreactivity of spiny projection neurons. Due to their large size and small number these cells most likely represent aspiny cholinergic interneurons. To verify this assumption, striatal cholinergic neurons were identified by the presence of choline [page 76↓]acetyltransferase (ChAT), the enzyme that catalyses the formation of acetylcholine from acetyl-CoA and choline.

Fig. 23: Kir2.4 channel immunoreactivity is most prominent in large striatal interneurons. Immunoreactivity for the Kir2.4 subunit in the rat striatum is mostly somatodendritic and largely absent from the neuropil (A). Staining is weak, however, in the large population of small striatal neurons and prominent in only a few, very large cells (A, arrows; B), most likely representing the so-called ‘giant’ interneurons in the striatum. Immunoreactivity is abolished (C) subsequent to preincubation of anti-Kir2.4 antibodies with the cognate protein.

Double labeling experiments (Fig. 24) confirmed the staining of cholinergic interneurons by the anti-Kir2.4 antibody. Under red fluorescence the microscopic image displays about five different neurons expressing Kir2.4 channel proteins (Fig. 24B, arrowheads). Green ChAT immunoreactivity presents a similar distribution of neurons (Fig. 24A). One single small cell, weakly stained for Kir2.4 (arrow in B), remained immunonegative for ChAT. The expression of Kir2.4 channels at cholinergic interneurons is verified by the yellow color of labeled neurons in the overlay image (Fig. 24C). The single smaller cell displays a red fluorescence only (Fig. 24C, arrow). Most probably it represents a spiny striatal projection neuron devoid of ChAT immunoreactivity. In addition to the Kir2.4 channel protein, the [page 77↓]Kir2.2 subunit was also localized to cholinergic interneurons, confirming data from in situ hybridization experiments (see discussion).

Fig. 24: Immunofluorescence double labeling of large striatal interneurons for choline acetyltransferase (ChAT) and Kir2.4 immunoreactivity. Sections were treated simultaneously with monoclonal mouse anti-ChAT- and rabbit anti-Kir2.4-antibodies, followed by visualization with Oregon Green-labeled goat anti-mouse and Texas Red-labeled goat anti-rabbit secondary antibodies. The four ChAT-positive cholinergic neurons (A) express Kir2.4-immunoreactivity (B, arrowheads) as judged from their yellowish appearance (C) after double exposition for green and red fluorescence.

The expression of Kir2 channels at ChAT neurons was confirmed at the electron microscopic level, also focusing on subcellular channel distribution. For this purpose cholinergic interneurons were labeled with anti-ChAT antibodies and visualized via peroxidase and diaminobenzidine. Anti-Kir2.2 and anti-Kir2.4 antibodies were detected using gold-labeled secondary antibodies. Both Kir2 subfamily members were localized at cholinergic interneurons (Fig. 25).


[page 78↓]

Fig. 25: Double labeling of Kir2.4 and choline acetyltransferase at the electron microscopic level. Cholinergic interneurons are identified by their amorphous diaminobenzidine reaction product (A, arrows) in single- (A) or double-stained sections. Kir2 immunoreactivity is visualized by silver-intensified ultrasmall gold particles (for details see methods). Cholinergic interneurons display Kir2.2 (B) as well as Kir2.4 (C) immunoreactivity.

DAB-positive cholinergic neurons were recognized by dark cytoplasmic deposits (Fig. 25A, arrows). In double labeling experiments, gold dots indicated the localization of Kir2.2 (Fig. 25B) and Kir2.4 (Fig. 25C) channel subunits. Note that there are some gold dots outside the cholinergic interneuron in anti-Kir2.2- but not in anti-Kir2.4-stained sections. Thus, only Kir2.4-immunoreactivity was found to be restricted to cholinergic inter-neurons, offering possibilities for specific, Kir2.4-based modulation of the striatal cholinergic system with potential therapeutic consequences.


Footnotes and Endnotes

1 Liu, G.X. 2001

2 Morishige, K.-I. 1993; Horio, Y. 1996; Karschin, C. 1999; Bredt, D.S. 1995; Töpert, C. 1998

3 Karschin, C. 1996

4 Inanobe, A. 2002

5 Morishige, K.-I. 1993; Horio, Y. 1996; Karschin, C. 1999; Bredt, D.S. 1995; Töpert, C. 1998

6 Karschin, C. 1996

7 Karschin, C. 1996

8 Töpert, C. 1998

9 Karschin, C. 1996; Karschin, C. 1999

10 Karschin, C. 1996

11 Horio, Y. 1996; Karschin, C. 1999

12 Töpert, C. 1998; Karschin, C. 1996

13 Karschin, C. 1999; Bredt, D.S. 1995

14 Falk, T. 1995

15 Stonehouse, A.H. 1999; Karschin, C. 1996

16 Parent, M. 2001

17 Pert, C.B. 1976



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