Unitary IPSPs evoked by interneurons at the stratum radiatum-stratum lacunosum-moleculare border in the CA1 area of the rat hippocampus in vitro

Abstract

1. Hippocampal non-principal neurons at the stratum radiatum-stratum lacunosum-moleculare border (R-LM interneurons) of the CA1 area may constitute several cell classes and have been implicated in the generation of GABAergic unitary IPSPs. Using biocytin-filled electrodes we recorded R-LM interneurons intracellularly in vitro and determined their postsynaptic effects in concomitantly recorded pyramidal cells. 2. Light microscopic analysis revealed four populations of R-LM interneurons with distinct axons: (1) basket cells (n = 4) with axons predominantly ramifying in the pyramidal cell layer; (2) Schaffer collateral/commissural pathway-associated interneurons (n = 10) stratifying in stratum radiatum and, to a lesser extent, stratum oriens; (3) perforant pathway-associated interneurons (n = 6) innervating the perforant path termination zone in stratum lacunosum-moleculare of the CA1 area as well as equivalent portions of the dentate gyrus and subiculum; and (4) neurogliaform interneurons (n = 2) characterized by their dense, compact axonal and dendritic arbour. 3. Random electron microscopic sampling of synaptic targets revealed a preponderance of pyramidal neurons as postsynaptic elements. Basket cells had a synaptic target preference for somata and proximal dendrites, whereas the remainder of R-LM interneurons innervated dendritic shafts and spines. The axon of dendrite-targeting cells formed up to six putative contacts with individual postsynatpic pyramidal cells. 4. Anatomically recovered R-LM interneurons (n = 22) had a mean resting membrane potential of -56.7 +/- 3.6 mV, a membrane time constant of 12.9 +/- 7.7 ms and an input resistance of 86.4 +/- 29.2 M omega. Depolarizing current pulses generally elicited overshooting action potentials (70.8 +/- 6.9 mV) which had a mean duration, when measured at half-amplitude, of 0.7 +/- 0.1 ms. In response to prolonged (> 200 ms) depolarizing current pulses all R-LM interneurons displayed (a varying degree of) spike frequency adaptation. 5. Basket cells, Schaffer-associated and neurogliaform interneurons elicited small-amplitude (< 2 mV), short-latency IPSPs in postsynaptic pyramids (n = 5, 13 and 1, respectively). Those interactions in which an effect was elicited with the repetitive activation of the presynaptic neuron (n = 13) showed a substantial degree of postsynaptic response summation. Unitary IPSPs had fast kinetics and, whenever tested (n = 5; 1 basket cell and 4 Schaffer-associated interneurons), were abolished by the GABAA receptor antagonist bicuculline. 6. Thus, R-LM interneurons comprise several distinct populations which evoke fast GABAA receptor mediated IPSPs. The domain-specific innervation of postsynaptic pyramidal cells suggests functionally diverse effects on the integration of afferent information in functionally non-equivalent compartments of pyramidal cells.

Figure 1. Synaptically connected basket-to-pyramidal cell pair in the CA1 area of the rat hippocampus

A, light microscopic reconstruction of the basket cell at the stratum radiatum-stratum lacunosum-moleculare border (soma and dendrites in red, axon in black; basket cell main axon denoted by arrow) and the synaptically coupled pyramidal cell (soma and dendrites in blue, spines and axon not shown). Ba, injection of a depolarizing current pulse (200 ms duration, 0.7 nA amplitude) into the basket cell elicited a train of accommodating action potentials. Bb, single basket cell action potentials evoked by depolarizing current pulses (0.25 nA) elicited short-latency, fast IPSPs in the concomitantly recorded pyramidal cell (Bc; average of 763 sweeps). Unitary IPSPs were hyperpolarizing events due to chloride loading of the pyramidal cell. The postsynaptic response was completely abolished by 10 μM of the bath applied GABA_A receptor antagonist bicuculline (Bd; average of 275 sweeps). C, amplitude histogram of unitary IPSPs (blue; n = 763) elicited at an average membrane potential of -71.6 ± 0.6 mV (mean ±s.d.). The corresponding noise distribution (black bars) was determined from the pre-event baseline. D, unitary IPSP amplitudes plotted as a function of the pyramidal cell membrane potential. Squares represent average amplitudes of 120-200 successive unitary IPSPs elicited at a given membrane potential. Linear regression was employed to extrapolate the IPSP response reversal (E_IPSP) and the resulting slope measurements (S_IPSP) were taken to calculate the unitary IPSP conductance (G_IPSP). Abbreviations: str. pyr., stratum pyramidale; str. rad, stratum radiatum; str. l-m., stratum lacunosum-moleculare.

Figure 2. A Schaffer-associated interneuron at the stratum radiatum-stratum lacunosum-moleculare border and two synaptically coupled pyramidal cells

A, light microscopic reconstruction of the biocytin labelled interneuron (soma and dendrites in red, axon in black; main axonal trunk denoted by arrow) and both postsynaptic pyramidal cells (P1 and P3; somata and dendrites in blue, spines and axons omitted). Ba, train of accommodating action potentials in response to a depolarizing current pulse (200 ms duration, 0.5 nA amplitude). Bb, brief bursts of action potentials in the presynaptic interneuron elicited small-amplitude summated IPSPs in both pyramidal cells (Bc and Bd; averages of 304 and 479 sweeps, respectively). C, light microscopically determined putative sites of synaptic interaction between the interneuron axon (black) and one of the pyramidal cells (P1, blue). All suspect sites (arrowheads, 1-6) were positioned on the shaft of small-calibre dendritic branches. Abbreviations: str. pyr., stratum pyramidale; str. rad., stratum radiatum; str. l-m., stratum lacunosum-moleculare.

Figure 3. Unitary IPSPs evoked by Schaffer-associated interneurons are mediated by GABA_A receptors

A, light microscopic reconstruction of a representative member of this interneuron class (soma and dendrites in red, axon in black) with a synaptically coupled pyramidal cell (soma and dendrites in blue, axon not shown). Ba, accommodating train of action potentials in response to injecting a depolarizing current pulse (200 ms duration; 0.3 nA amplitude) into the interneuron. Brief trains of 4-5 presynaptic action potentials (Bb - single sweep) elicited incrementally summating IPSPs in the pyramidal cell. Bc, the postsynaptic response remained unaltered in the presence of bath applied glutamate receptor antagonists (10 μM CNQX and 50 μM AP5) but was virtually eliminated due to the successive application of the GABA_A receptor antagonist bicuculline (Bd; 10 μM). Be, following a 20 min wash-out with bicuculline-free ACSF partial response recovery became apparent, with the unitary IPSP amplitude returning to ≈70 % of control levels. Bf, superimposition of averages shown in c-e. C, graphic reconstruction showing all 6 light microscopically determined sites of presumed synaptic interaction (arrowheads, 1-6) between the interneuron axon (in black; only partially displayed) and the postsynaptic pyramidal cell (in blue). Abbreviations: str. pyr., stratum pyramidale; str. rad., stratum radiatum; str. l-m., stratum lacunosum-moleculare.

Figure 7. Dendritic geometry of interneurons at the stratum radiatum-stratum lacunosum-moleculare border

Based on their axonal distribution pattern (here not shown; for cells 2, 5, 6, 10 and 13 of Table 2, see Figs 1, 2, 3, 5 and 6, respectively), all cells could be incorporated into four major classes. Although differences in the axonal distribution pattern were generally more striking and sufficient for categorization, there appeared to be several group-specific distinguishing features in the dendritic architecture of stratum radiatum-stratum lacunosum-moleculare interneurons. Basket cell dendritic arbours (3/4) were radially elongated and spanned all hippocampal layers. Dendritic trees of Schaffer- and perforant path-associated interneurons had a more stellate appearance, with dendrites only infrequently entering stratum oriens (ori.) and the alveus (alv.). Both neurogliaform interneurons had very small stellate dendritic arbours. Abbreviations: l-m., stratum lacunosum-moleculare; rad., stratum radiatum; pyr., stratum pyramidale.

Figure 4. Summation and decay of unitary IPSPs mediated by Schaffer-associated interneurons

Aa, presynaptic trains of 7 action potentials evoked in an interneuron (single sweep) elicited short-latency IPSPs in a simultaneously recorded pyramidal cell (Ab; average of 334 sweeps). Initially several postsynaptic responses show a substantial degree of response summation (coupling artifacts denoted by arrows) until a plateau is reached. The IPSP decay could be adequately fitted with a single exponential. Ba, similar to the example shown in A, tetanic activation of a different Schaffer-associated interneuron (single sweep) elicited incrementally summating IPSPs in a concomitantly recorded pyramidal cell (Bb, average of 110 postsynaptic responses). As in panel Ab the IPSP decayed monoexponentially.

Figure 5. Properties of a perforant path-associated interneuron

A, light microscopic reconstruction of the interneuron (soma and dendrites in red, axon in black, main axon denoted by short arrow) having an axonal arbour not only within stratum lacunosum-moleculare (str. l-m.), but also in the outer portions of the dentate gyrus molecular layer (DG m.l.) and adjoining parts of the subiculum (CA1-subicular border denoted by stippled line). A long arrow denotes the position of an additional collateral which (re)crossed the hippocampal fissure and gave rise to a large axonal plexus in the presubiculum (not shown). B, firing pattern of the perforant path-associated interneuron in response to a 200 ms duration 0.7 nA depolarizing current pulse. The cell displayed prominent spike frequency accommodation and a slow after-hyperpolarizing potential. Abbreviations: str. pyr., stratum pyramidale; str. rad., stratum radiatum; g.c.l., granule cell layer; hilus, hilar region of dentate gyrus.

Figure 6. Anatomy and postsynaptic effect of a neurogliaform interneuron at the stratum radiatum-stratum lacunosum-moleculare border

A, light microscopic reconstruction of the interneuron (soma and dendrites in red, axon in black, main axon denoted by arrow). Ba, a train of weakly accommodating action potentials evoked by a depolarizing current pulse (100 ms duration; 0.5 nA amplitude) in the neurogliaform interneuron. Bb, single action potentials in the presynaptic interneuron elicited small-amplitude fast IPSPs in a simultaneously recorded pyramidal cell (Bc; average of 140 sweeps). Due to the brief recording time the pyramidal cell was morphologically not recovered. Abbreviations: str. pyr., stratum pyramidale; str. rad., stratum radiatum; str. l-m., stratum lacunosum-moleculare.

Figure 8. Electron micrographs of synaptic junctions established by identified interneurons at the stratum radiatum-stratum lacunosum-moleculare border

A-J, labelled boutons were followed through serial ultrathin sections until they formed synaptic junctions (arrows) with their respective postsynaptic targets (data shown in Table 2). A, basket cell (2806951; cell 4 of Table 2) bouton forming synaptic contacts (one each) with neighboring pyramidal somata (S). The right-most synaptic junction is clearly identifiable, whereas the synaptic cleft of the junction shown to the left is more readily apparent after tilting the specimen (not shown). B and C, consecutive sections of a synaptic bouton of a Schaffer-associated interneuron (1201965; cell 6 of Table 2, shown in Fig. 3A). This terminal established two synaptic junctions with a large (D₁) and a small-calibre dendritic shaft (D₂). D, vesicle-filled terminal of a second Schaffer-associated interneuron (1805951; cell 5 of Table 2; shown in Fig. 2A) in synaptic contact with the shaft of a non-principal dendrite (D), also receiving converging synaptic input (arrowheads). E, a synaptic junction established by a perforant pathway-associated interneuron (1505951; cell 12 of Table 2) on a granule cell dendrite (D) in the outer molecular layer of the dentate gyrus. F and G, a second perforant pathway-associated interneuron (2706951; cell 11 of Table 2) formed synaptic junctions on medium to small-calibre dendritic shafts of pyramidal cells (D), here at the origin of spines (asterisks). The spine in F received a converging asymmetrical synapse (open triangle). H-J, randomly encountered synaptic junctions made by neurogliaform cells (H is cell 0107951, cell 14 of Table 2; I and J are cell 2606951, cell 13 of Table 2, see Fig. 6A). Neurogliaform cells formed rather inconspicuous synaptic junctions primarily within stratum radiatum (H and J) and, less commonly, in stratum lacunosum-moleculare (I). Scale bars: 0.25 μm. Bar in A also applies for E and I; bar in B also applies for C, D and G; bar in F also applies for H.