Morphology and Physiology of Cortical Neurons in Layer I

CR cells

CR cells are thought to be the first postmitotic cells that appear during embryonic development (Marin-Padilla, 1984; Bayer and Altman, 1990; Derer and Derer, 1990; Huntley and Jones, 1990). Biocytin labeling from cells in slices obtained from 7- to 9-d-old rats revealed CR-like cells (n = 6; Fig. ​Fig.11,C1,C2). These neurons were characterized by an orientation largely horizontal to the one to three primary processes that arose from a round-to-ovoid soma (16.3 ± 4.5 × 10.3 ± 2.5 μm). Four of the six cells were bipolar in appearance, with two processes extending laterally from each pole of the soma. One process was thick, with a roughly contoured appearance resulting from a high density of appendages. These appendages varied from small spine-like protrusions to longer filiform processes. Often the thicker process expanded distally. From the opposite somatic pole, the opposing process was thin and axon-like, as in Figure ​Figure2.2. Both thick and thin processes could give rise to thin, vertical branchlets, which in turn could branch and project horizontally (Fig. ​(Fig.2).2). These thin processes extended anteroposteriorly several hundred micrometers.

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Fig. 2.

The projection tube drawing of a biocytin-stained CR cell (same cell as in Fig. ​Fig.1)1) from a 9-d-old animal. Thearrowhead indicates the possible site of axon origin. Note the location of the cell in the upper portion of layer I (indicated by the bracket at right of the drawing). Note also the thick single dendrite with small finger-like appendages. Inset, A response to an injection of depolarizing current. Note slow action-potential time course and progressive increase in spike width. Resting potential was −50.5 mV.

The remaining two CR cells had processes with a similar morphology except that the bipolar orientation was not evident. Regardless, a thin process arose directly from either the soma or a thick process and projected several hundred micrometers beneath the pial surface, branching frequently. All of the CR cells were located in the upper half of layer I. In older rats (11–19 d), we recovered no cells with the unique CR morphology.

The average resting potential of CR cells was −64.4 ± 8 mV, and all CR cells were electrically active, as action potentials could be initiated with depolarizing current (Fig. ​(Fig.2,2, inset). These spikes had a long duration (half-width: 3.9 ± 0.7 msec). Trains of spikes were not always induced and when present were characterized by pronounced spike broadening and amplitude reduction. The input resistance of CR cells was high (610 ± 163 MΩ).

Neurogliaform cells

Eight of the 26 cells we recorded in slices from 11- to 19-d-old rats had a very dense axonal arbor that was contained almost entirely within layer I, and these were characterized as neurogliaform cells (Figs. ​(Figs.11A1,A2, ​,3).3). In addition to the dense local axonal arbor, neurogliaform cells had five to six smooth and short primary dendrites that were restricted to layer I, and a small smooth soma (15.7 ± 4.3 × 9.9 ± 1.9 μm). Distally, all of the processes, but particularly the axon, were very thin and required high-magnification, oil-immersion optics to be followed. Neurogliaform cells were typically located in the middle to upper half of layer I. The axonal projection field could extend throughout the dorsoventral extent of the layer and anteroposteriorly 200–300 μm and could fill the thickness of the slice. The density of the arborization and the thinness of the processes often made distinguishing axons from dendrites difficult.

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Fig. 3.

A projection tube drawing of a biocytin-stained neurogliaform cell in layer I. Borders of layer I are indicated atleft of the drawing. The extensive axonal arbor extends close to the border of layer I but does not enter layer II. The central location of the somata is typical of these cells. Inset, A single action potential initiated in this cell by current injection (300 pA, 10 msec) is followed by an afterdepolarization (ADP). Resting potential: −56 mV.

The average resting potential of neurogliaform cells was −62.0 ± 6.8 mV, and their average input resistance and membrane time constant were 500 ± 166 MΩ and 71 ± 42 msec (n = 8), respectively. Current injections from resting potential elicited single spikes at threshold level (Fig.​(Fig.44A). Action potentials had a short half-width (1.2 ± 0.2 msec) and a large fast afterhyperpolarization potential (fAHP), measured as the difference between spike threshold and the peak hyperpolarization immediately after the spike (−19.9 ± 2.3 mV). In response to suprathreshold current injection, spike trains were often interrupted by quiescent periods (Fig. ​(Fig.44B). Larger current injection resulted in an uninterrupted train of spikes showing some spike frequency adaptation in all but one neuron, which responded with only one spike. In addition to these characteristics, we also found that seven of the eight neurogliaform neurons tested exhibited a slow membrane depolarization after action potentials (+5.8 ± 1.7 mV) (Figs. ​(Figs.33,inset, ​,44B–D). This afterdepolarization (ADP) peaked at 60.1 ± 28.7 msec after the termination of a single spike or a short train of action potentials and repolarized slowly with a time constant of 203 ± 60.2 msec (Figs. ​(Figs.3,3, inset,​,44B–D). The ADP was observed after a train of spikes (Fig.​(Fig.44C,D) or could be initiated by a single spike (Fig. ​(Fig.33,inset; see Fig. ​Fig.88D). The ability of cells to generate an ADP was not strongly dependent on the resting membrane potential. Under membrane hyperpolarization to −80 mV or more negative, however, the rising phase of the ADP was obscured by the spike repolarization. As will be shown below, the current underlying the ADP is inward at the resting membrane potential. Also shown in Figure ​Figure44A is a depolarizing sag that developed with stronger membrane hyperpolarization, which was found to some degree both in neurogliaform cells and in cells with descending axons.

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Fig. 4.

Action-potential characteristics of a neurogliaform cell. All records are from the same cell.A, Steps (900 msec) of current injection (−30, −10, +10, +50, and +70 pA) from resting potential (−65 mV). Note that the response to hyperpolarizing current injection (−30 pA) suggests the presence of an inward rectification, as indicated by the sag (open circle). At threshold, a single spike was evoked with a large fAHP. Note the PSPs (arrows) indicated by the fast rise and slow decay with a peak of ∼1 mV. B, A depolarizing current injection above threshold evoked a single spike followed by a quiescent period preceding further spikes. Note the prolonged depolarization after current cessation. C, Suprathreshold current injection evokes a train of spikes with little frequency adaptation. Note the ADP after current injection (filled circle). D, ADP (filled circle) initiated by short-duration current injection. Resting potential: −65 mV.

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Fig. 8.

AHP and ADP. A, AHP after a single action potential in a cell with a descending axon. Note the PSPs at the end of the voltage trace. Resting potential: −65 mV; current injection: +200 pA, 10 msec. B, ADP in a neurogliaform cell induced by a short train of spikes. Note the small PSP occurring at the decay phase of ADP. Resting potential: −62 mV; current injection: +200 pA, 50 msec. Current injection: C andC1, AHP and I-AHP in a single cell. I-AHP is induced by a brief (10 msec) depolarizing pulse of membrane potential to +20 mV from a holding potential of −60 mV. Note that the I-AHP (C1) has a time course similar to the AHP (C). D and D1, ADP and I-ADP in a single neurogliaform cell. Under current clamp (D), depolarizing current (+300 pA, 10 msec) induced a single spike from a resting potential of −61 mV. I-ADP was induced by a 2 msec voltage step to 20 mV from a holding potential of −65 mV (D1). Note the slow rising phase of the response under voltage clamp.

Cells with a descending axon

Seven of the 26 cells from 11- to 19-d-old rats had an axon that descended to layer II or deeper (Figs. ​(Figs.11B1,B2,​,5).5). Like neurogliaform cells, the somata were small (14.9 ± 3.4 × 7.9 ± 1.7 μm) but were typically found in the middle to lower half of layer I. These cells had four to five sparsely branching dendrites. Two of the seven neurons had spiny dendrites, whereas the remaining five were relatively aspiny. For most of these neurons, the dendritic tree extended from the top to the bottom of layer I; additional dendritic projections were commonly found in the upper half of layer II. The anteroposterior extent of dendritic spread, however, was limited to <200 μm from either side of the soma. The distal portions of the dendrites were thin, as were secondary and tertiary branches. The single axon arose from either the soma or a primary dendrite. Although the axon sometimes branched within layer I, the local arborization was sparse when compared with that of neurogliaform cells. Scattered axonal branches were also found in deeper layers, and varicosities were often visible along collateral branches. The axon of two neurons reached layer IV and that of a third reached layer V. The remaining neurons had axons restricted to layers I and II. Collateral branches in deeper layers were not observed to extend anteroposteriorly much beyond ∼100 μm from the cell body. The axons were thin distally, however, and the staining was faint, and we cannot rule out a more extensive projection.

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Fig. 5.

Cell with a descending axon. Soma is located close to layer I–II border (indicated at left of drawing). The axon, whose origin is indicated by arrowhead, descends well into lower layers but gives off a local arbor.Inset, A train of spikes is followed by an afterhyperpolarization (AHP). Resting potential: −59 mV; current injection: 150 pA, 200 msec.

The resting potential (−63.8 ± 5.1 mV), input resistance (756 ± 308 MΩ), and membrane time constant (79 ± 27 msec) of cells with a descending axon were not significantly different from those of neurogliaform cells. These cells responded with a single spike to threshold stimulation and under suprathreshold current injection fired a steady train of action potentials. Action potentials had a half-width of 1.7 ± 0.3 msec and an fAHP of −12.2 ± 1.7 mV. Compared with neurogliaform cells, the fAHP was smaller (p ≤ 0.02), whereas the action-potential half-width was longer (p ≤ 0.02). During a spike train, there was spike frequency adaptation (Fig.​(Fig.66A), and action potentials were broadened (Fig. ​(Fig.7).7). Cells with a descending axon exhibited more frequency adaptation than did neurogliaform cells. The ratio of the spike frequency of the fourth to the first interspike interval from briefly evoked spike trains was 0.85 ± 0.1 versus 0.70 ± 0.14 in neurogliaform cells and cells with descending axons, respectively (p ≤ 0.05). After a train of spikes, cells with a descending axon exhibited an AHP (Fig.​(Fig.66B2). An AHP was also induced by a single spike (Fig.​(Fig.66B1). The average amplitude and decay time constant of the AHP were −5.1 ± 2.7 mV and 211.6 ± 44.0 msec, respectively. Because of the more extensive spike broadening exhibited by cells with descending axons, the differences in the half-width and fAHP between these and neurogliaform cells were accentuated when the second spike was examined, and together these two features clearly distinguished the two cell types (Fig. ​(Fig.7).7). A single cell with a descending axon displayed a notch after the action potential, and an action potential could be triggered after a rebound from hyperpolarization (data not shown).

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Fig. 6.

Action potential characteristics of cells with descending axon. A, Depolarization induced a train of action potentials illustrating spike frequency adaptation. Note the spike broadening during the train. After the train there is an AHP (indicated by filled circle). Resting potential: −59 mV; current injection: +100 pA, 700 msec. Inset, Superimposition of the first (arrowhead) and the twelfth (arrow) spikes of the train. Note the broadening of the action potential. Spike width at half amplitude was 1.7 msec (first) and 3.4 msec (twelfth). The spikes were aligned at the threshold. Scale is 2 msec, 10 mV. B1, B2, A different cell exhibiting an AHP after a single spike (B1) or a short train of spikes (B2). Resting potential: −62 mV; current injection: 220 pA, 10 msec (B1) and 120 pA, 100 msec (B2). The AHPs are indicated by filled circles.

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Fig. 7.

Action potential parameters in neurogliaform cells and cells with descending axons. The fAHP plotted against the half-width of the second spike in a train of spikes. ThefAHP of neurogliaform cells (filled symbols) is larger in amplitude than that of cells with descending axons (open symbols). The second spike half-width of the neurogliaform cell is shorter in duration compared with that of cells with descending axons. Inset, Superimposition of the first and second spikes in a train of action potentials. Inset left, Neurogliaform cell; inset right, cell with descending axon.

Other neurons

Three neurons were recovered that had an axon apparently restricted to layer I but did not locally arborize with the density of neurogliaform cells and thus were not classified as such. In addition, some of the dendrites extended into layer II. None of these three cells exhibited an ADP, and only one exhibited the slow AHP characteristic of cells with descending axons. Soma size averaged 16.8 ± 1.4 × 9.1 ± 0.5 μm.

The remaining neurons from which we recorded physiological data and recovered a stained neuron were left unclassified because the axon was not well stained. These cells were morphologically and electrophysiologically diverse; two exhibited an ADP and had short dendrites restricted to layer I, reminiscent of neurogliaform cells; five had a slow AHP and longer dendrites that often extended into layer II. Soma size ranged from 15.2 × 2.6 μm for the largest to 10.4 × 7.2 μm for the smallest neuron.

Voltage clamp

The slow time course of the ADP and AHP displayed by the neurogliaform cells and cells with a descending axon, respectively, may reflect slowly relaxing voltage-dependent currents or may be driven by calcium or other second messengers. Under current-clamp conditions, the time course of the ADP or AHP depends on voltage-dependent conductances and the membrane time constant and therefore may not represent the kinetics of the underlying conductance waveform. Figure​Figure88 illustrates AHPs and ADPs in cells with a descending axon (Fig. ​(Fig.88A,C) and in neurogliaform cells (Fig.​(Fig.88B,D), respectively. Under voltage clamp, a brief membrane depolarization induced a slowly developing outward current in neurons with an AHP (Fig. ​(Fig.88C,C1) and an inward current in cells with an ADP (Fig. ​(Fig.88D,D1). The decays of the I-AHP and I-ADP had a time course of several hundred milliseconds, similar to that of the voltage traces after action potentials. Furthermore, the I-ADP had a clear rising phase, suggesting that this current does not represent a tail current but reflects a slow response to events triggered by brief membrane depolarization.

The AHP has been observed in several cell types and is generated by an increase in K-conductance (for review, see Sah, 1996). The mechanisms underlying the ADP, however, have not been established. It has been suggested that in pyramidal and nonpyramidal neurons the ADP may reflect a calcium tail current, a decrease of potassium conductance (Constanti et al., 1993), an electrogenic ion pump (Friedman et al., 1992), or a nonselective cationic conductance (Caeser et al., 1993). We measured the voltage dependency of the conductance underlying the ADP by stepping the membrane potential to various voltages at the peak of the I-ADP (Fig. ​(Fig.99A). The tail-current amplitude, defined as the difference between the steady-state current at the end of the trace and the current just after the voltage step plotted against the membrane voltage, indicated a reversal potential near −50 mV (Fig. ​(Fig.99B). These data suggest that the conductance mechanism is most likely an increase in mixed cationic conductance rather than a decrease in potassium conductance or calcium tail current.

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Fig. 9.

Conductance mechanism of the I-ADP.A, The membrane potential was −65 mV. A brief (2 msec) voltage pulse to −20 mV initiated the I-ADP. At the peak of the I-ADP, the membrane potential was stepped to different voltages: −75, −65, −60, −55, −50, and −45 as shown diagrammatically beneath the current traces. Note that the tail current after a step to −75 mV was larger than that observed at more depolarized voltages, indicating that the reversal potential was more depolarized than −75 mV.B, The current–voltage relationship of the tail currents was obtained by subtracting the current (averaged over 5 msec) measured at the time indicated by the right open arrowfrom the current just after the voltage step at the time indicated by the left open arrow. Note that the tail current reversed polarity at approximately −50 mV. Same cell as that shown in Figure​Figure88D.

Neurogliaform cells

Neurogliaform cells have been described by several authors (Jones, 1984). Ramon y Cajal (1911) reported neurogliaform cells in layer I that he also called spiderweb or dwarf cells. Neurogliaform cells have been described in layer I of human newborn infants (Marin-Padilla, 1984) and in the rat visual cortex (Hedlich and Werner, 1987). Martin et al. (1989) used intracellular recording and HRP injection to obtain the receptive field properties and morphology of a single, layer I neuron. The morphology of that cell is similar to that of the neurogliaform cells we describe here (compare Fig. ​Fig.22 of Martin et al., 1989, and Figs. ​Figs.11 and ​and22 in this paper). Sousa-Pinto et al. (1975),Winer and Larue (1989), and Anderson et al. (1992) have not identified neurogliaform cells in layer I, but this may reflect incomplete staining of the fine axons. The robust filling of neurogliaform cells we have obtained in some cells revealed a very dense axonal arbor that rarely extended beyond the border of layer I. These data suggest that neurogliaform cells synapse primarily onto targets within layer I. These targets could include the dendrites of pyramidal neurons, dendrites from nonpyramidal neurons of layer II/III (Kawaguchi, 1995), and other layer I neurons. Kawaguchi (1995) described neurogliaform cells in layer II/III of frontal cortex after biocytin filling, with morphological characteristics remarkably similar to those reported herein.

It has been shown previously that spiking parameters can be used to differentiate pyramidal from nonpyramidal cortical neurons (McCormick et al., 1985; Connors and Gutnick, 1990). Further differentiation among cortical nonpyramidal neurons in layers II–VI has also been observed (Foehring et al., 1991; Kawaguchi, 1993, 1995, 1996; Kawaguchi and Kubota, 1993). The electrophysiological properties of neurogliaform cells have been studied only by Kawaguchi (1995), who found that these cells exhibit a delayed spike occurring at the end of a depolarizing current pulse. We have not observed the late spiking behavior in layer I neurogliaform cells, which possibly reflects different conditions of our experiments or perhaps a difference between layer I and layer II/III neurogliaform cells. We found, however, that layer I neurogliaform cells exhibit a characteristic ADP after spikes, which was not reported by Kawaguchi (1995). Cortical pyramidal neurons also exhibit a form of ADP, the time course of which is typically faster than the ADP in neurogliaform cells (Friedman and Gutnick, 1987;Schwindt et al., 1988; Foehring and Waters, 1991). Under voltage clamp, brief depolarization of neurogliaform cells generated a slow inward current (I-ADP) with kinetics similar to those of the ADP, suggesting that the time course of the ADP is not reflecting the membrane time constant but rather a slow conductance. The reversal potential of approximately −50 mV suggests a mixed cationic conductance. A cationic conductance is thought to underlie the ADP of hippocampal pyramidal cells induced by activation of glutamate metabotropic and cholinergic muscarinic receptors (Caeser et al., 1993).

In response to a steady current injection, layer I neurogliaform cells fire action potentials characterized by a large fAHP and less frequency adaptation and spike broadening when compared to cells with descending axons. Spike frequency adaptation and spike broadening are prominent in most pyramidal neurons and are found to a lesser degree in some nonpyramidal cells (Kawaguchi, 1995, 1996).

Cells with descending axons

We have identified a group of cells that had a prominent descending axon in addition to their local collateral projections. The characteristics of cells with a descending axon have not been studied extensively in the literature; however, Ramon y Cajal (Fig. 54 inDeFelipe and Jones, 1988) described layer I cells, which he called cells with descending axons, that had been discovered previously by Schaffer in 1897 (cited in DeFelipe and Jones, 1988). He described them as being located in the lower part of layer I, and they seem to correspond to the cells with descending axons that we describe here.

It is not possible to determine from our material whether the axons extend to the white matter, because the axonal staining became faint distally. Martínez-García et al. (1994) found that some cells in the inner half of layer I project to the contralateral visual cortex. Therefore, it may be that some of the cells we classify as cells with descending axons provide input to distant regions in addition to the collateral inputs seen in deeper cortical layers.

Cells with a descending axon are clearly differentiated from neurogliaform cells both morphologically and electrophysiologically. Compared with neurogliaform cells, those with a descending axon exhibit a smaller fAHP and a larger spike width. The slow AHP and frequency adaptation seen in cells with a descending axon recall the regular spiking nonpyramidal cells described by Kawaguchi (1995; 1996) in layers II/III and V. Cells with a descending axon are morphologically similar to the subclass of nonpyramidal neurons that are immunoreactive for vasoactive intestinal polypeptide (Kawaguchi, 1996).

CR cells

CR cells are among the first postmitotic cells. Recent findings suggest that they are important developmentally (Ogawa et al., 1995); however, their fate in the adult cortex has not been resolved. Only a few authors have reported on CR cells in the adult brain (Condé et al., 1994; Martínez-García et al., 1994). Indeed, we have identified CR cells only in relatively young animals (7- to 9-d-old). Presently it is not known whether CR cells die at early postnatal age, change their morphology, or diminish in proportion to other proliferating cells (Marin-Padilla, 1984; Bayer and Altman, 1990; Derer and Derer, 1990; Huntley and Jones, 1990; del Rio et al., 1995). Electrophysiologically, CR cells are active, as has been shown recently by Zhou and Hablitz (1996) (Fig. ​(Fig.2,2, inset), and their spike parameters are similar to immature cortical neurons (Kriegstein et al., 1987; McCormick and Prince, 1987; Lorenzon and Foehring, 1993; Kim et al., 1995; Zhou and Hablitz, 1996).

Unclassified cells

Our reticence to classify approximately one third of the neurons reflects what we consider to be weak or incomplete staining of axonal arbors. In addition, the morphologies of a few well filled neurons simply could not be generalized to other groups of neurons and stood as isolated examples. It is likely that layer I contains other distinct groups of neurons that we have not identified simply because they were insufficiently represented in our study.