The dentate gyrus is known for its laminar inputs with commissural and associational fibers targeting the inner molecular layer (IML) while medial and lateral perforant path project to the middle and outer molecular layers (Frotscher, 1991). Inhibitory projections share a similar laminar structure with parvalbumin-expressing fast-spiking basket cell (FS-BC) axons in the granule cell layer, hilar commissural-associational pathway-associated (HICAP) cells and CCK-expressing neurons projecting to the IML and somatostatin-expressing hilar-perforant pathway-associated (HIPP) cells terminating in the outer molecular layer (Buckmaster et al., 2002; Hefft and Jonas, 2005). Additionally, there exists a class of total molecular layer cells (TML-Cs) with axonal projection across the entire dentate molecular layer (Soriano and Frotscher, 1993; Buckmaster and Schwartzkroin, 1995; Mott et al., 1997). TML-Cs have high axonal density in the middle molecular layer and are positioned to mediate feedback inhibition of medial perforant path inputs (Soriano and Frotscher, 1993; Hosp et al., 2014). Although described as relatively numerous (Mott et al., 1997; Zhang and Buckmaster, 2009), little is known about TML-Cs aside from morphology and basic firing characteristics (Soriano and Frotscher, 1993; Mott et al., 1997; Hosp et al., 2014). Determining the neurochemical identity and synaptic characteristics of TML-Cs is essential to defining their role in the dentate circuit.

A related intriguing issue concerns the expression of cannabinoid receptor type 1 (CB₁R) in the dentate molecular layer. CB₁R is present in certain glutamatergic and GABAergic terminals in the dentate IML (Monory et al., 2006; Morozov et al., 2009). Among glutamatergic terminals, CB₁R is present exclusively in mossy cell axons and excluded from perforant path inputs (Monory et al., 2006). Although axons of CCK-positive neurons known to express CB₁R are localized to the IML, deletion of CB₁R from glutamatergic terminals reveals CB₁R labeled axons distributed across the entire molecular layer (Monory et al., 2006). The axonal distribution of TML-Cs raises the possibility that these neurons may have CB₁R-positive axons and undergo cannabinoid-modulation of synaptic release. This study was conducted to identify the synaptic features and CB₁R expression profile of dentate TML-Cs in order to aid further analysis of their contribution to circuit function.

Briefly, horizontal brain slices (300 μM) were prepared from male, Wistar rats >30 days old under protocols approved by Rutgers-NJMS, Newark, NJ, IACUC. Neurons in the subgranular hilus were patched using microelectrodes containing equal concentrations of KCl and K-gluconate and 0.2% biocytin (Proddutur et al., 2013; Yu et al., 2013). Intrinsic properties were determined from responses to 1.5 sec current injections from a -70 mV holding potential. Only neurons with dendrites in the hilus and molecular layer and total molecular layer axons on post-hoc morphological analysis were included as TML-Cs (Soriano and Frotscher, 1993). Neurons with non-adapting high frequency firing with axons in the granule cell layer and co-labeling for parvalbumin were considered FS-BCs (Yu et al., 2013). Neurons with axons predominantly in the IML and adapting firing pattern were considered HICAP cells. For paired recordings, presynaptic interneurons were stimulated with 2−8 current pulses (3 ms, 700-1100pA) at 50 Hz every 10 s in current-clamp mode while postsynaptic neurons were voltage-clamped at −70 mV. Synaptic events were analyzed using Clampfit (Molecular Devices). Following recordings, slices were fixed 4% paraformaldehyde and immunolabeled with anti-CCK (1 : 1,000, monoclonal mouse, courtesy of G. Ohning, CURE, UCLA), anti-CB₁R (1 : 1,000, polyclonal guinea pig, Frontier Science) or anti-PV antibody (1.5 : 1,000, polyclonal rabbit, Swant) using previously described protocols (Yu et al., 2013). Some experiments included resectioned or perfusion-fixed sections (50 μm). Biocytin staining was revealed using Alexa 594-conjugated streptavidin. Images were obtained using a Nikon A1R laser confocal microscope (1.2 NA 60X water objective) and used for morphological reconstruction (Neurolucida). Data are presented as mean ± s.e.m. P < 0.05 by t-test or Mann-Whitney U test (for data that failed normality test) were considered significant.

TML-Cs were identified by the relatively sparse axonal distribution in the dentate molecular layer with occasional hilar collaterals (Figs. 1A and 2A), as shown before (Soriano and Frotscher, 1993; Buckmaster and Schwartzkroin, 1995; Mott et al., 1997). Consistent with previous reports (Soriano and Frotscher, 1993), TML-Cs had relatively small somata (247.8 ± 27.2 μm², n = 12 cells) compared to FS-BCs (350.8 ± 19.3 μm², n = 12 cells, P < 0.05) and aspiny dendrites extending into both the molecular layer and hilus. Molecular layer dendrites of TML-Cs extended to the hippocampal fissure (Fig. 1A). Intrinsic physiology of TML-Cs was characterized by accommodating firing pattern, distinct from the non-adapting firing of FS-BCs (Fig. 1B). TML-Cs had lower maximum discharge frequency (Fig. 1C, frequency in Hz at 800 pA current injection 36.2 ± 5.5, n = 14 cells) and higher input resistance (R_in) (Figs. 1B,D, R_in in MΩ, 222.6 ± 17.1, n = 14 cells) than FS-BCs (frequency in Hz: 112.1 ± 7.5, n = 12 R_in in MΩ, 93.0 ± 10.1, n = 12 cells, reported in Proddutur et al., 2013; Yu et al., 2013). Additionally, unlike FS-BCs, TML-Cs showed spike frequency adaptation (Figs. 1B,E, Ratio ISI_first/last, TML-C: 0.33 ± 0.04, n = 14 cells, FS-BC: 0.84 ± 0.04, n = 12 cells) during sustained depolarization and a greater membrane potential sag during hyperpolarization (Sag ratio, TML-C: 0.86 ± 0.02, n = 14 cells, FS-BC: 0.96 ± 0.01, n = 12 cells, P < 0.05 by t-test). Although TML-C intrinsic properties such as adapting firing pattern, high input resistance and presence of membrane sag resemble those of CCK expressing HICAP cells (Mott et al., 1997; Savanthrapadian et al., 2014), they were morphologically distinguished from HICAP cells based on axonal distribution in the middle and outer molecular layers.

In paired interneuronal recordings, TML-C activation evoked monosynaptic responses in hilar interneurons including TML-Cs (Figs. 2A,B, n = 5 pairs) and FS-BCs (n = 7 pairs, not shown). Compared to synapses between FS-BCs (7 pairs), unitary inhibitory postsynaptic currents (uIPSCs) between TML-Cs (5 pairs) were characterized by low release probability (Figs. 2B,C; success rate, TML-C: 22 ± 3%; FS-BC: 86 ± 6%, P < 0.05) and amplitude (Figs. 2B,D; uIPSC amplitude including failures in pA, TML-C: 4.4 ± 1.2; FS-BC: 185 ± 98.8, P < 0.05 by U test). TML-C uIPSC amplitude potency excluding failures was 19.5 ± 3.9 pA. Similarly, 20–80% rise time (in ms, TML-C: 0.96 ± 0.19; FS-BC: 0.39 ± 0.05, P < 0.05 by U test) and decay times (τ_decay in ms, TML-C: 8.28 ± 2.08; FS-BC: 3.23 ± 0.61, P < 0.05 by U test) were slower than in FS-BC synapses. TML-C synapses had longer latency (Fig. 2E; in ms, TML-C: 1.8 ± 0.2, FS-BC: 0.8 ± 0.1, P < 0.05 by U test) and higher CV of latency (TML-C: 0.41 ± 0.14, FS-BC: 0.14 ± 0.02, P < 0.05 by U test) than FS-BCs, which is similar to data from IPSCs between presumed HICAP cells (Savanthrapadian et al., 2014). Moreover, TML-C synapses showed paired and multi-pulse facilitation (Figs. 2B,G) rather than the depression between FS-BCs (Savanthrapadian et al., 2014). TML-Cs exhibit both synchronous and asynchronous release in response to activation at 50 Hz (Fig. 2G), which is similar to the asynchronous release reported in HICAP cells and in CCK-expressing neurons in the hippocampus (Ali and Todorova, 2010; Savanthrapadian et al., 2014; Szabo et al., 2014). Synaptic responses from TML-Cs were blocked by 10 μM SR95531 (n = 3 pairs, not shown) indicating that inhibition was mediated by GABA_A receptors. These data demonstrate that, like intrinsic properties, synaptic characteristics of morphologically identified TML-Cs are distinct from FS-BC and similar to dentate HICAP cells (Savanthrapadian et al., 2014). To our knowledge, these data represent the first functional characterization of TML-C synapses.

Since TML-Cs show asynchronous synaptic release characteristic of CB₁R-expressing neurons, we examined if TML-Cs express CB₁R. Immunostaining for CB₁R and CCK in dentate sections revealed distinct CB₁R positive fibers in the middle and outer molecular layers that did not co-localize with CCK (not shown), in addition to IML axonal fibers co-labeled for CB₁R and CCK. Since perforant path inputs do not exhibit cannabinoid modulation (Chancey et al., 2014) and CB₁R-positive mossy cell axons are restricted to the IML (Monory et al., 2006), our immunostaining data suggest that interneurons with axons spanning the molecular layer likely express CB₁R. Direct examination of TML-Cs filled with biocytin during recordings revealed somatic labeling for CB₁R (Figs. 3A–D, reconstruction of the cell in Figure 1A confirms TML-C morphology, n = 11 of 11 cells tested). However, TML-Cs lacked somatic or dendritic labeling for CCK (Figs. 3A–D, n = 8) despite prominent CCK immunoreactivity in adjacent neurons (Fig. 3C, inset panels to right of 3D show colocalization of CB₁R in the CCK expressing cell). Since TML cells showed somatic labeling for CB₁R, and neurons with IML axons recorded under similar conditions were co-labeled for CCK and CB₁R (data not shown), it is unlikely that the recording conditions resulted in absence of CCK expression in TML-Cs. Thus, TML-Cs are morphologically distinct from HICAP cells and can be neurochemically distinguished from CCK- and CB₁R-positive neurons with IML axons. In addition to the soma, molecular layer axon collaterals of TML-Cs were labeled with CB₁R (Figs. 3E–J, panels E-G are from cell in 3A, H, I, and J are from two different cells) indicating that TML-Cs may contribute to the expansive CB₁R labeling in dentate GABAergic terminals (Monory et al., 2006; Magloczky et al., 2010).

Activation of CB₁R, in the presynaptic terminals of CCK-positive interneurons, leads to a reduction in probability of synaptic release from CCK neurons to principal cells and interneurons (Freund, 2003; Armstrong and Soltesz, 2012). The characteristic baseline and activity-dependent cannabinoid modulation of synaptic release has been used to distinguish between inhibition from CCK-positive interneurons and FS-BCs that lack cannabinoid modulation. Given the expression of CB₁R on TML-C axons we examined whether TML-C synapses show CB₁R-dependent modulation synaptic release. In 4 out of 4 TML-C pairs tested, the CB₁R antagonist, AM251 (10 μM) consistently and reversibly enhanced the synaptic success rate (Figs. 4A,B, 194.2 ± 12.0% of baseline, 4 pairs) and uIPSC amplitude (Figs. 4A,B, 195.0 ± 29.8% of baseline, 4 pairs). In two TML-C pairs tested, the CB₁R agonist WIN-55212 (10 μM) reduced the probability of synaptic release (from 0.23 to 0.13, and from 0.2 to 0.1 respectively) suggesting that CB₁R-modulation of TML-C synaptic release is bidirectional. Additionally, release probability at TML-C synapses on FS-BCs was also enhanced by AM251 (4 pairs, not shown). Together these data demonstrate functional, baseline endocannabinoid modulation of unitary TML-C synapses.

This study constitutes the first detailed physiological and neurochemical characterization of dentate TML cells, a class of GABAergic neurons in the hilar-granule cell layer border. Although TML-Cs were initially described over 20 years ago (Soriano and Frotscher, 1993) and have been observed during dentate interneuronal recordings (Mott et al., 1997; Zhang and Buckmaster, 2009; Hosp et al., 2014) their synaptic properties have not been examined. Apart from synaptic contacts on presumed granule cells (Soriano and Frotscher, 1993), we find that TML-Cs innervate other interneurons including TML-Cs and FS-BCs. Functionally, TML-C synapses show high temporal jitter in latency and asynchronous release suggesting that TML-Cs, like CCK-positive cells, contribute to prolonged synaptic inhibition during sustained network activity rather than the temporally precise inhibition of FS-BCs (Freund, 2003; Armstrong and Soltesz, 2012; Savanthrapadian et al., 2014). Moreover, the low reliability and multi-pulse facilitation also favor a more sustained role for TML-Cs in dentate inhibition. While we directly demonstrate CB₁R-sensitive synapses between TML-Cs, release at TML-C synapses on granule cells and other interneurons are also likely modulated by CB₁Rs. Thus, in addition to the commissural-associational inputs in the IML that are regulated by asynchronous inhibition from HICAP cells, perforant path inputs in the middle and outer molecular layers are potentially regulated by feed-forward and feedback inhibition from TML-Cs with facilitating synaptic dynamics.

A salient finding of this study is that TML-Cs, show somatic and axonal expression of CB₁R. TML-C synapses exhibit low release probability that was enhanced by the CB₁R inverse agonist AM251 (Fig. 4B) indicating baseline CB₁R-dependent suppression of release as reported in cortical and hippocampal CB₁R-expressing neurons (Losonczy et al., 2004; Foldy et al., 2006). The presence of CB₁R and not CCK in TML-Cs is distinct from both HICAP cells expressing CCK designated as CCK-positive basket cells (Hefft and Jonas, 2005; Savanthrapadian et al., 2014) and HICAP cells lacking CB₁R-modulation (Liu et al., 2014). Moreover, unlike hippocampal neurons that co-localize CCK and CB₁R (Ali and Todorova, 2010; Lee et al., 2010; Szabo et al., 2014), TML-Cs uniquely express CB₁R and lack CCK. While earlier studies have suggested the presence of CB₁R in dentate interneurons lacking CCK (Hajos et al., 2000; Morozov et al., 2009), our study provides the first morphological and functional identification of these neurons. By demonstrating that neurons with dentate molecular layer axons express CB₁R and not CCK, our findings shed light on differential distribution of CCK- and CB₁R-expressing inhibitory axonal collaterals across the dentate molecular layer (Hefft and Jonas, 2005; Magloczky et al., 2010; Monory et al., 2006). More importantly, our data demonstrate that CB₁R-sensitive inhibition in the dentate cannot be considered synonymous with inhibition from CCK-expressing interneurons.