Reply to Liu and Bilkey's reply

Reply to Liu and Bilkey’s Reply

L. Stan Leung and Kevin J. Canning

Liu and Bilkey found a peak current sink at the hippocampal fissure afterperirhinal cortex (PRh) stimulation. One of our contentions with their datais that the location of sink was not accurately determined. The localizationof a sink at the distal dendritic layers of CA1 and/or the dentate gyrusrequires a level of accuracy that was lacking in Liu and Bilkey’s studies(1996, 1997). In their reply, Liu and Bilkey confirmed that theirlocalization of the sink at the hippocampal fissure was inferred from thedepths of the CA1 and dentate granule cell (GC) layers, and the cell layerswere detected by audio monitoring (of unit activity). This method of depthestimation is not accurate. The hippocampal fissure is about 250 and 500mm from the GC and CA1 cell layer, respectively. Liu and Bilkey’s estimatewill be subjected to large uncertainties introduced by distortion of the tissueby lowering their 60-mm electrode, shrinkage of tissue after histologicalprocessing, and the errors in precisely determining the cell layers. Tominimize these errors, we advocate the method of depositing a smallamount of dye from a glass micropipette at the depth of interest, in this caseat the distal dendrites of CA1 (see our letter above).

Liu and Bilkey replied that their sink ‘‘peak occurred at the fissure, as onewould expect if there were current sinks located both above and below thisregion.’’ As an analytical tool, current source density (CSD) analysis shouldbe used to determine where the sinks are actually located, independent ofexpectation or the anatomical data. In our studies on hippocampalresponses following medial or lateral perforant path stimulation (Leung etal. 1995; Canning and Leung 1997), we reported that the dendritic sinktransients at the dentate gyrus and CA1 have different electrophysiologicalproperties, such as threshold stimulus intensity, onset and peak latencies,and paired-pulse facilitation. Unfortunately, Liu and Bilkey have not shownthat their fissure sink was resolvable into two different components, eitherin location or in properties. However, they believe that both CA1 anddentate gyrus generated the sink. Given that a dentate sink was much morereadily activated than a CA1 sink following PRh stimulation, it is likely thatLiu and Bilkey had activated only one sink in the dentate gyrus. The lattersink was accompanied by a source in the dentate gyrus.

We propose that a sink in CA1 was not activated by Liu and Bilkey alsobecause they found no CA1 source accompanying the CA1 sink. Contraryto what Liu and Bilkey suggested, this source in CA1 does not depend onstimulus intensity or smoothing technique. Low stimulus intensity at themedial perforant path may not evoke a CA1 distal dendritic sink, but whenthe sink was found, a mid-dendritic source in CA1 was also found.Smoothing by different formulas (D1, D2, or D4 were discussed in ourletter above) may affect the spatial decay, magnitude, and extent of thesource, but not the existence of the source itself. The presence of the source,simultaneous with the sink, comes from the biophysical principle that aninflow of current at one part of the cell must be matched by an outflow ofcurrent at another part of the cell. Thus, current source-sink dipolesunderlie the generation of extracellular potentials and monopoles in theCSD do not exist. The finding of only sink and no source (or vice versa)may suggest that the electrode track is not appropriately placed. Either the

track has not passed through the regions that generatethe currents, or it is oblique to the main flow of currents.In either case, the basic assumption of one-dimensionalCSD analysis is violated and the CSD data must bequestioned (see, e.g., Leung, 1979). Liu and Bilkeycommented that they ‘‘often failed to observe a source’’in CA1 following stimulation of the lateral perforantpath (LPP); perhaps CA1 was not activated in theseexperiments either (see Canning and Leung, 1997).

Liu and Bilkey drew our attention to their findingthat the amplitude of a LPP to hippocampal responsewas diminished by ibotenic acid lesion of the PRh andnot by electrolytic lesion of the lateral entorhinal cortex(LEC). The preservation of LPP to hippocampal re-sponses after LEC lesion may suggest that the part ofLEC they lesioned did not project axons near their LPPstimulating electrode, which is of dubious relevance tothe PRh projection issue. Functional plasticity (e.g.,long-term potentiation) or structural reorganization ofthe remaining LEC may also contribute to the LPPresponse in the lesioned animals. In contrast to theabolition of LPP-to-hippocampal response after ibotenicacid lesion of the PRh, we have emphasized the preserva-tion of hippocampal responses evoked by a stimulatingelectrode in the PRh after extensive cell death in the PRh(Canning and Leung, 1997). From the latter data, wehave concluded that the response evoked by PRhstimulation in a normal rat mainly resulted fromstimulation of fibers of passage and not perirhinalcortical neurons.

We have also argued that anatomical evidence doesnot favor Liu and Bilkey’s conclusion of a perirhinal toCA1/dentate gyrus projection. The lack of anatomicalevidence of a perirhinal to dentate pathway has beendiscussed in the Letter and Reply of Witter et al. (thisissue). The projection of the PRh to the CA1/subiculumborder is limited to a small region around P5.8 in the rat,as shown by Kosel et al. (1983) and McIntyre et al.(1996), and now confirmed by Naber et al. (in press).There is no evidence of a perirhinal to CA1 projection atthe level P3.8 in the rat, where Liu and Bilkey foundtheir CA1 distal dendritic sink.

Our conclusions remain the same. We believe that aPRh to CA1 projection is not substantiated by theelectrophysiological data of Liu and Bilkey, and a PRh todentate gyrus projection is not supported by anatomicaldata or our physiological study (Canning and Leung,1997).

HIPPOCAMPUS 9:603–604 (1999)

r 1999 WILEY-LISS, INC.

REFERENCES

Canning KJ, Leung LS. 1999. Current source density analysis does notreveal a direct projection from the perirhinal cortex to septal part ofhippocampal CA1 or dentate gyrus. Hippocampus 9:599–600.

Canning KJ, Leung LS. 1997. Lateral entorhinal, perirhinal, andamygdala-entorhinal transition projections to hippocampal CA1 anddentate gyrus in the rat: a current source density study. Hippocampus7:643–655.

Kosel KC, Van Hoesen GW, Rosene DL. 1983. A direct projection fromthe perirhinal cortex (area 35) to the subiculum in the rat. Brain Res269:347–351.

Leung LS, Roth L, Canning KJ. 1995. Entorhinal inputs to hippocampalCA1 and dentate gyrus in the rat: a current-source-density study. JNeurophysiol 73:2392–2403.

Leung LS. 1979. Potentials evoked by the alvear tract in hippocampalCA1 of rats. II. Spatial field analysis. J Neurophysiol 42:1571–1589.

Liu P, Bilkey DK. 1996. Direct connection between perirhinal cortex andhippocampus is a major constituent of the lateral perforant path.Hippocampus 6:125–134.

Liu P, Bilkey DK. 1997. Current source density analysis of the potentialevoked in hippocampus by perirhinal cortex stimulation. Hippocam-pus 7:389–396.

McIntyre DC, Kelly ME, Staines WA. 1996. Efferent projections of theanterior perirhinal cortex in the rat. J Comp Neurol 369:302–318.

Naber PA, Witter MP, Lopes da Silva FH. Perirhinal cortex input to thehippocampal formation in the rat: evidence for parallel pathways,both direct and indirect. Eur J Neurosci, in press.

Witter MP, Naber PA, Lopes da Silva FH. 1999. The perirhinal cortexdoes not project to the dentate gyrus. Hippocampus 9:605–606.

Witter MP, Naber PA, Lopes da Silva FH. 1999. Perirhimal cortex doesnot project to the dentate gyrus. Hippocampus 9:605–606.

604 REPLY TO