Characterization of neurons in the CA2 subfield of the adult rat hippocampus

Abstract

The hippocampal cornu ammonis 2 (CA2) region is unique in being the only CA region receiving inputs from the hypothalamic supramammillary nucleus, of importance in modulating hippocampal theta rhythm, and is seizure resistant in temporal lobe epilepsy. CA2 has, however, been little studied, possibly because of its small size and difficulty encountered in defining its borders. To investigate the properties of CA2 interneurons, intracellular recordings with biocytin filling were made in adult hippocampal slices. Two types of basket cells were identified. A minority resembled those in CA1, with fast spiking behavior, vertically oriented dendrites, and axons confined to the region of origin. In contrast, the majority of parvalbumin-immunopositive CA2 basket and bistratified cells had long, horizontally oriented, sparsely spiny dendrites extending into all CA subfields in stratum oriens, adapting firing patterns and a pronounced "sag" in voltage responses to hyperpolarizing current, indicative of I(h). Broad CA2 basket cells innervated all three CA subfields and could thus provide CA1 and CA2 with feedforward and CA3 with feedback inhibition. In contrast, CA2 bistratified cell axons displayed striking subfield preference, innervating stratum oriens and stratum radiatum of CA2 and CA1 but stopping abruptly at the CA2/CA3 border, implying feedforward inhibition of CA2 and CA1. These unique features suggest that CA2 is more than a transitional region between CA1 and CA3. The pronounced slow sag current of many CA2 interneurons may contribute to coordination of pyramidal cell firing during theta, whereas the fast spiking behavior of a smaller population of interneurons supports more localized gamma.

Figure 1.

A, B, Characterization of the CA2 region using Timm staining (A) and immuno-peroxidase technique labeling the α-actinin 2 protein (B). Sections 50 μm thick were cut from an ∼2500 μm tissue block containing the hippocampus. Superimposition of the two stains is shown in C, demonstrating that the mossy fibers taper into the CA2 region as defined by the α-actinin 2 staining.

Figure 2.

Morphological features of pyramidal cells recorded and filled in the CA1 and CA2 regions of the adult rat hippocampus. A, B, Neurolucida reconstructions of CA2 pyramidal cells. Like those in CA1, the dendritic arbors of CA2 pyramidal cells (black) extended through SO and SLM. Their axons (gray) arborized either exclusively in SO (A) or in SO and SR of the CA2 and CA3 regions (B). C, Neurolucida reconstruction of a typical CA1 pyramidal cell. The dendritic arbor (black) extended through all layers, and the axon (gray) emitted collaterals in SO. D, Number of dendritic branches against the distance from the soma of CA1 and CA2 pyramidal cells. All data are represented as mean ± SD. E, Length of apical and basal dendrites of CA1 and CA2 pyramidal cells. CA2 pyramidal cells had longer basal and shorter apical dendrites than CA1 pyramidal cells (Mann–Whitney U test, p < 0.05; U_basal = 3, U_apical = 0; n_CA1 = 6, n_CA2 = 6).

Figure 3.

Dendritic and axonal arbors of basket cells recorded and filled in the CA1 and CA2 regions. Aa, Reconstruction of a CA1 basket cell (drawing tube, 1000×). The dendrites of CA1 basket cells (in black) extended through SO and SLM. The axonal arbor (in red) was almost entirely confined to SP of the CA1 region. Ab, The basket cell was PV immunopositive and CB immunonegative. Ac, Image of a portion (black square in Aa) of a distal dendrite that was beaded and aspiny. Ba, Reconstruction using a drawing tube (1000×) of a CA2 basket cell that was confined to the CA2 region. The dendrites are in black, and the axon is in red. Bb, Image of a portion (black square in Ba) of a distal dendrite that was beaded and aspiny. Bc, Superimposition of the drawing of the basket cell on an image of the α-actinin 2 staining confirmed that the interneuron was confined to the CA2 region. Ca, A CA2 basket cell was reconstructed using a drawing tube (1000×). The dendritic tree of this basket cell (in black) extended radially through all layers of the CA2 region and horizontally in SO and SP of the CA2 and CA3 regions. One horizontal dendrite also reached the CA1 region. The axon (in red) extended to the CA3 and CA1 regions. Cb, The basket cell was PV immunopositive and CB immunonegative. Cc, Spines (arrows) were present in the distal part of the horizontally oriented dendrites (black square in Ca). Cd, Superimposition of the drawing of the basket cell on an image of the α-actinin 2 staining.

Figure 4.

Dendritic and axonal patterns of bistratified cells recorded and filled in the CA1 and CA2 regions. Aa, Reconstruction of a typical bistratified cell in the CA1 region. The dendritic arbor (in black) extended radially through SO and SR but did not enter SLM. The axon (in red) arborized mainly in SO and SR of the CA1 region. Ab, The bistratified cell was PV immunopositive and CB immunonegative. Ac, Image of a portion (black square in Ba) of a distal dendrite that was beaded and aspiny. Ba, Reconstruction of a CA2 bistratified cell using a drawing tube. This cell presented a narrow dendritic arbor (in black) that extended from SO to SR without entering SLM. The axon (in red) was strongly polarized and arborized mainly in SO and SR of CA2 and CA1 but did not enter CA3. The bistratified cell was CCK immunonegative. Bb, Image of a portion (black square in Ba) of a distal dendrite that was beaded and aspiny. Bc, Superimposition of the drawing of the bistratified cell on an image of the α-actinin 2 staining. Ca, Reconstruction of another CA2 bistratified cell. This interneuron presented a broad dendritic arbor (in black) that extended to the CA1 and CA3 regions. The axonal arbor of this bistratified cell (in red) was strongly polarized ramifying in SO and SR of the CA2 and CA1 regions but not CA3. Cb, Image of a portion (black square in Ca) of a distal dendrite that presented spines (arrows). Cc, Superimposition of the drawing of the bistratified cell on an image of the α-actinin 2 staining.

Figure 5.

Electrophysiological characteristics of basket cells recorded in the CA1 and CA2 regions. A, Voltage responses of a CA1 basket cell to hyperpolarizing and depolarizing current injection. B, Voltage responses of a CA2 basket cell that had a narrow dendritic arbor. C, Voltage responses of a CA2 basket cell that had broad and horizontally oriented dendrites. In contrast to CA1 basket cells and to CA2 basket cells that were confined to CA2, this cell displayed significant spike frequency adaptation and a pronounced sag in its voltage response to hyperpolarizing current injection.

Figure 6.

Electrophysiological characteristics of bistratified cells recorded in the CA1 and CA2 regions. A, Voltage responses of a CA1 bistratified cell to hyperpolarizing and depolarizing current injection. B, Voltage responses of a CA2 bistratified cell that had a narrow dendritic arbor. C, Voltage responses of a CA2 bistratified cell that had broad and horizontally oriented dendrites. In contrast to CA1 and CA2 bistratified cells with narrow dendritic arbors, this cell displayed significant spike frequency adaptation and a pronounced sag in its voltage response to hyperpolarizing current injection.

Figure 7.

Correlation between the width of horizontally oriented dendrites and the presence of a sag in the voltage responses to hyperpolarizing current injection. All basket and bistratified cells with broad dendritic arbors displayed a sag in their voltage response to a current pulse of −0.6 nA (Spearman's test, r = 0.74; α = 0.05).