PA Schwartzkroin

Multiple voltage-gated potassium (K) channel gene products are likely to be involved in regulating neuronal excitability of any single neuron in the mammalian brain. Here we show that two closely related voltage-gated K channel proteins, mKv1.1 and mKv1.2, are present in multiple subcellular locations including cell somata, juxta-paranodal regions of myelinated axons, synaptic terminals, unmyelinated axons, specialized junctions among axons, and proximal dendrites. Staining patterns of the two channel polypeptides overlap in some areas of the brain, yet each has a unique pattern of expression. For example, in the hippocampus, both mKv1.1 and mKv1.2 proteins are present in axons, often near or at synaptic terminals in the middle molecular layer of the dentate gyrus, while only mKv1.1 is detected in axons and synaptic terminals in the hilar/CA3 region. In the cerebellum, both channel proteins are localized to axon terminals and specialized junctions among axons in the plexus region of basket cells. Strong differential staining is observed in the olfactory bulb, where mKv1.2 is localized to cell somata and axons, as well as to proximal dendrites of the mitral cells. This overlapping yet differential pattern of expression and specific subcellular localization may contribute to the unique profile of excitability displayed by a particular neuron.

Electrophysiological and anatomical techniques were used to determine the role, in the hippocampal circuitry, of local circuit neurons located at the oriens/alveus border (O/A interneurons). Intracellular recording from these cells showed that their response characteristics were clearly nonpyramidal: high input resistance, short membrane time constant, short-duration action potential, pronounced, brief afterhyperpolarizations (AHP), and nondecremental firing during intrasomatic depolarizing current pulses. Intracellular Lucifer yellow (LY) injection and subsequent fluorescence microscopy confirmed their nonpyramidal nature. O/A interneuron somata were bipolar or multipolar; their dendrites projected mostly parallel to the alveus, except for 1 or 2 processes that turned perpendicularly, and ascended through stratum oriens and pyramidale and into radiatum. Their axons were seen to branch profusely in stratum oriens and pyramidale. Simultaneous intracellular recordings from O/A interneurons and CA 1 pyramidal cells showed that pyramidal cells directly excite these interneurons. Major hippocampal afferents also directly excited the O/A interneurons. In a small number of interneuron-pyramidal pairs, stimulation of the O/A interneuron directly inhibited pyramidal cells. In one case, reciprocal connections were observed: The pyramidal cell excited the interneuron, and the interneuron inhibited the pyramidal cell. In 1 interneuron-to-interneuron pair, an inhibitory connection from O/A interneuron to stratum pyramidale interneuron was also observed. With intracellular HRP injections into O/A interneurons and subsequent electron microscopy, we observed that O/A interneuron axons made contacts with pyramidal and nonpyramidal cells. HRP-filled symmetric synaptic contacts were found on pyramidal cell dendrites and somata. HRP-filled axons also made contacts with pyramidal cell initial segments. HRP-filled O/A interneuron axon contacts were also found on nonpyramidal cell dendrites in stratum oriens. These electrophysiological and anatomical results suggest that O/A interneurons make synaptic contact with pyramidal cells and may mediate feedforward and feedback inhibition onto CA 1 pyramidal cells.

The tumor suppressor gene p53 recently has been associated with the induction of cell death in response to some forms of cellular damage. A possible role for p53-related modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. We evaluated the possibility that p53 expression (in knockout mice) is required for induction of cell damage in a model of seizure activity normally associated with well defined patterns of cell loss. Subcutaneous injection of kainic acid, a potent excitotoxin, induced comparable seizures in both wild-type mice (+/+) and mice deficient in p53 (-/-). Using a silver impregnation technique to examine neurodegeneration in animals killed 7 d after kainate injection, we found that a majority of +/+ mice exhibited extensive cell loss in the hippocampus, involving subregions CA1, CA3, the hilus, and the subiculum. Apoptotic cell death, as identified with an in situ nick end labeling technique to detect DNA fragmentation, was confirmed in CA1- but not CA3-degenerating neurons. In marked contrast, a majority of p53 -/- mice displayed no signs of cell damage; in the remaining p53 -/- mice, damage was mild to moderate and was confined almost entirely to cells in CA3b of the dorsal hippocampus. In +/+ mice, but not in -/- mice, damaged neurons also were observed in the amygdala, piriform cortex, cerebral cortex, caudate-putamen, and thalamus after kainate treatment. The pattern and extent of damage in mice heterozygous for p53 (+/-) were identical to those seen in +/+ mice, suggesting that a single copy of p53 is sufficient to confer neuronal vulnerability. These results demonstrate that p53 influences viability in multiple neuronal subtypes and brain regions after excitotoxic insult.

The hypothesis that recurrent inhibition in the hippocampus is mediated by interneurons was tested with simultaneous intracellular recordings from the CA1 region of guinea pig hippocampal slices in vitro. In recordings from 101 pairs of pyramidal cells, no interactions were detected in 87% of the pairs. In 13% of the pyramidal cell pairs, spike trains induced in one cell caused inhibitor postsynaptic potentials (IPSPs) in the second cell. No excitatory interactions were detected. In recordings from 43 pairs of cells, where one cell was a pyramidal cell and the other cell was an interneuron, no interactions were detected in 53% of the pairs. In 30% of the interneuron-pyramidal cell pairs, spike trains elicited from the interneuron caused IPSPs in the pyramidal cell. In 28% of the pairs, spike trains elicited from the pyramidal cell caused excitatory postsynaptic potentials (EPSPs) in the interneurons. In 4% of these pairs, reciprocal interactions were seen, with the pyramidal cell exciting the interneuron and the interneuron inhibiting the pyramidal cell. These results support the hypothesis that inhibitory mediate recurrent inhibition in the hippocampus. However, the data also suggest that the interneurons from which these results were recorded were a subset of inhibitory interneurons distinct from the classical basket cell. These interneurons may mediate both feed-forward and recurrent inhibition in the hippocampus.

Stable intracellular recordings were obtained from nonpyramidal cells (interneurons) in stratum lacunosum-moleculare (L-M) of the CA1 region of guinea pig hippocampal slices. The intracellular response characteristics of these interneurons were distinctly different from responses of pyramidal cells and of other interneurons (basket cells and oriens-alveus interneurons). L-M interneurons had a high resting membrane potential (-58 mV), a high input resistance (64 M omega), and a large amplitude (60 mV), relatively long duration (2 msec) action potential. A large afterhyperpolarization (11 mV, 34 msec) followed a single action potential. Most L-M interneurons did not display any spontaneous firing. Lucifer yellow (LY)-filled L-M interneurons showed nonpyramidal morphology. Cells were generally fusiform or multipolar, with aspinous, beaded dendritic processes ramifying in stratum lacunosum-moleculare, radiatum, and (sometimes) oriens. The varicose axon originated from a primary dendrite, projected along stratum lacunosum-moleculare, branched profusely in stratum radiatum, and coursed toward and into stratum pyramidale and occasionally into oriens. Processes of cells with somata in the L-M region of CA1 were not restricted to the CA1 region. The dendritic and axonal processes of some L-M interneurons were seen ascending in stratum lacunosum-moleculare, crossing the hippocampal fissure, and coursing in stratum moleculare of the dentate gyrus. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were evoked in L-M interneurons from stimulation of major hippocampal afferents. EPSPs were most effectively elicited by stimulation of fiber pathways in transverse slices, whereas IPSPs were predominantly evoked when major pathways were stimulated in longitudinal slices. We have identified a population of interneurons with intracellular response characteristics and morphology distinctly different from previously described pyramidal and nonpyramidal neurons of CA1 region. The possible role of these interneurons in hippocampal circuitry is discussed.