Bidirectional Regulation of Cognitive and Anxiety-like Behaviors by Dentate Gyrus Mossy Cells in Male and Female Mice

Abstract

The dentate gyrus (DG) of the hippocampus is important for cognition and behavior. However, the circuits underlying these functions are unclear. DG mossy cells (MCs) are potentially important because of their excitatory synapses on the primary cell type, granule cells (GCs). However, MCs also activate GABAergic neurons, which inhibit GCs. We used viral delivery of designer receptors exclusively activated by designer drugs (DREADDs) in mice to implement a gain- and loss-of-function study of MCs in diverse behaviors. Using this approach, manipulations of MCs could bidirectionally regulate behavior. The results suggest that inhibiting MCs can reduce anxiety-like behavior and improve cognitive performance. However, not all cognitive or anxiety-related behaviors were influenced, suggesting specific roles of MCs in some, but not all, types of cognition and anxiety. Notably, several behaviors showed sex-specific effects, with females often showing more pronounced effects than the males. We also used the immediate early gene c-Fos to address whether DREADDs bidirectionally regulated MC or GC activity. We confirmed excitatory DREADDs increased MC c-Fos. However, there was no change in GC c-Fos, consistent with MC activation leading to GABAergic inhibition of GCs. In contrast, inhibitory DREADDs led to a large increase in GC c-Fos, consistent with a reduction in MC excitation of GABAergic neurons, and reduced inhibition of GCs. Together, these results suggest that MCs regulate anxiety and cognition in specific ways. We also raise the possibility that cognitive performance may be improved by reducing anxiety.SIGNIFICANCE STATEMENT The dentate gyrus (DG) has many important cognitive roles as well as being associated with affective behavior. This study addressed how a glutamatergic DG cell type called mossy cells (MCs) contributes to diverse behaviors, which is timely because it is known that MCs regulate the activity of the primary DG cell type, granule cells (GCs), but how MC activity influences behavior is unclear. We show, surprisingly, that activating MCs can lead to adverse behavioral outcomes, and inhibiting MCs have an opposite effect. Importantly, the results appeared to be task-dependent and showed that testing both sexes was important. Additional experiments indicated what MC and GC circuitry was involved. Together, the results suggest how MCs influence behaviors that involve the DG.

Keywords: contextual fear conditioning; hilus; immediate early gene; memory; novelty; object recognition.

Figure 1.

Experimental design. A, Viral constructs used for (A1) gain-of-function (eDREADD) and (A2) loss-of-function (iDREADD) experiments. B1, Schematic of the hippocampus. B2, 160 nl of virus was injected into the rostral and caudal hippocampus, bilaterally. C, Representative viral expression in rostral and caudal coronal sections of control and DREADD-injected mice. Scale bar, 200 µm. GCL: granule cell layer, IML: inner molecular layer, Hil: hilus D, Quantification of viral expression along the septotemporal axis. D1, Quantification of hilar cells expressing mCherry shows robust labeling. eDREADD and iDREADD mice were similar, so they are pooled in D2 and D3. D2, Fluorescence intensity in the IML suggests consistent expression. Y axis: 0 black, 255 white. D3, Measurement of the span of fluorescence in the IML shows similarity of lower and upper blades (eDREADD and iDREADD mice pooled). E, Simplified MC circuit diagram. (1) MCs excite GCs through a monosynaptic “direct” pathway. (2) MCs also inhibit GCs through an “indirect” MC→GABAergic neuron→GC inhibitory pathway. The indirect inhibitory pathway is thought to dominate the direct excitatory pathway under normal conditions.

Figure 2.

CFC in control, eDREADD, and iDREADD mice. A, Mice were placed in a fear conditioning chamber, and 3 footshocks (0.5 mA) were delivered 1 min apart. B, Minute-by-minute analysis of the training session found no effect of treatment on baseline freezing (B1,B2) or freezing during the first 2 PS minutes (PS1 and PS2). The eDREADD mice froze significantly less than controls in the third PS minute (PS3; p = 0.017) and less than control and iDREADD mice in the fourth minute (PS4; all p values < 0.011). C, When data were averaged across all PS minutes, eDREADD mice froze significantly less than control and iDREADD mice (all p values < 0.037). D, Female eDREADD mice froze significantly less than female control mice (p = 0.036), and female iDREADD mice had a similar pattern (p = 0.052). There was a sex difference in training, with female mice freezing significantly more than male mice (p < 0.001). Also, there was no significant effect of treatment in the male cohort. E, Mice were returned to the same fear conditioning apparatus 24 h later to assess fear memory. F, Minute-by-minute analysis of the first 5 min of the context test showed that eDREADD mice froze less than iDREADD (all p values < 0.047) and control mice (all p values < 0.033). G, When freezing behavior was averaged across the entire test duration, eDREADD mice showed significantly less freezing than control and iDREADD mice (all p values < 0.011). H, There was a significant effect of treatment in the female cohort, whereby eDREADD mice froze significantly less than control and iDREADD mice (all p values < 0.043). There was no effect of treatment in the male cohort. Data are mean ± SEM. *p < 0.05. B, F, *p < 0.05, significantly different from control. ^#p < 0.05, iDREADD significantly different from eDREADD.

Figure 3.

NOR in control, eDREADD, and iDREADD mice. A, In NOR training, mice explored two identical novel objects for 5 min. B, There was no effect of treatment on the training DI when both sexes were pooled. C, There was no effect of treatment on training DI in male and female cohorts. D, There was no effect of treatment on the total time spent exploring objects (A + B) during NOR training in female and male cohorts. E, Female iDREADD mice spent significantly less time exploring objects than female eDREADD mice during NOR training (p = 0.044). F, Male mice did not differ by treatment on time spent exploring Object A versus Object B during training. G, Mice were tested for object recognition memory 1 h after training by replacing Object B with a novel object. H, iDREADD mice had a significantly greater testing DI than eDREADD mice (p = 0.013). I, Testing DI did not differ by treatment in female mice. However, male control and iDREADD mice had a significantly greater testing DI than eDREADD mice (all p values < 0.034). J, Female and male mice did not differ by treatment in total object exploration during testing. K, There was no effect of treatment in female mice on the time spent exploring Object A versus Object B during testing (all p values > 0.065). L, Male iDREADD mice spent significantly more time exploring Object B than Object A during testing (p = 0.008). Error bars = SEM. *p < 0.05.

Figure 4.

NOL in control, eDREADD, and iDREADD mice. A, In NOL training, mice explored two identical novel objects for 5 min. B, The overall NOL training DI did not differ by treatment. C, There was no effect of treatment on NOL training DI in the female and male cohorts. D, Female and male mice did not differ in total object exploration time (Object A + Object B) during training. E, F, Female and male mice did not differ by treatment in the time spent exploring Object A versus Object B during training. G, Mice were tested for object location memory 1 h later by moving Object B to the other side of the testing arena. H, There was no significant effect of treatment on the testing DI. I, The testing DI did not differ by treatment in male and female cohorts. J, Female and male mice did not differ in their total object exploration time during testing. K, L, There was no effect of treatment in female and male mice on spent time spent exploring Object A versus Object B during testing. Error bars = SEM.

Figure 5.

HCNOE in control, eDREADD, and iDREADD mice. A, Two identical novel objects (yellow Legos, outlined in black; see arrows) were placed in the home cage. Object exploration was measured over the first 4 min. B, There was an overall effect of treatment on object exploration, with iDREADD mice spending a greater percent of time exploring objects than control and eDREADD mice (all p values < 0.019). Furthermore, eDREADD mice spent less time exploring objects compared with control mice (p = 0.010). C, There was a significant effect of treatment in the female cohort, with iDREADD mice spending a greater percent of time exploring than control and eDREADD mice (all p values < 0.039). Also, male iDREADD mice spent a greater time exploring objects than male eDREADD mice (p = 0.003). D, Minute-by-minute analysis found that iDREADD mice spent a greater percent of time exploring than eDREADD mice for each of the 4 min (all p values < 0.001) and greater exploration than control mice for the first 2 min (all p values < 0.017). Control mice also showed a greater percent of exploration than eDREADD mice during the fourth minute (p = 0.005). E, F, Minute-by-minute exploration in female and male mice. Overall, similar effects were observed as in the pooled analysis shown in D. Thus, iDREADD mice generally showed greater exploration than eDREADD mice and controls were often between the two treatment groups. D–F, Error bars = SEM. *p < 0.05, Significantly different from control. ^#p < 0.05, iDREADD significantly different from eDREADD.

Figure 6.

NSF in control, eDREADD, and iDREADD mice. A, Mice were food-deprived for 24 h and water-deprived for 2 h before undergoing the NSF test. Mice were placed in the corner of a brightly illuminated novel arena (X), and the latency to eat a food pellet in the arena was measured. B, There was a significant effect of treatment, with iDREADD mice eating ∼30% sooner than the eDREADD mice (p = 0.015). There were no other treatment differences in latency to feed. C, Female iDREADD mice had a significantly shorter latency to feed compared with control mice (p = 0.033). No other significant treatment differences were found between female mice (all p values > 0.054). The latency to feed did not differ between treatments in male mice. Error bars = SEM. *p < 0.05.

Figure 7.

LDB in control, eDREADD, and iDREADD mice. A, iDREADD mice spent ∼25% more time in the light compartment of the LDB compared with control mice (p = 0.036). B, There was no effect of treatment in female mice on the amount of time spent in the light compartment of the LDB. However, male iDREADD mice spent more time in the light compartment of the LDB compared with male control mice (p = 0.037). C, Representative heat maps of male (C1) control, (C2) eDREADD, and (C3) iDREADD mice in the light compartment of the LDB. The heat map calibration is not as precise as the quantitation in A, B and is for illustrative purposes only. D, E, There was no effect of treatment on the distance traveled in the light compartment of the LDB when subjects were pooled or separated by sex. Error bars = SEM. *p<0.05.

Figure 8.

OFT in control, eDREADD, and iDREADD mice. A, DREADD treatment had no significant effect on the amount of time spent in the center of the OFT. B, Female mice spent significantly less time in the center of the OFT compared with males (p = 0.011). C, Representative track map for female (C1) control, (C2) eDREADD, and (C3) iDREADD mice. Blue and red circles represent the start and end of the track path, respectively. D, There was no difference in the total distance traveled during the OFT. E, There was no difference in the total distance traveled during the OFT in female and male cohorts. Error bars = SEM. *p<0.05.

Figure 9.

EPM in control, eDREADD, and iDREADD mice. A, eDREADD mice spent a greater percent of time in the open arms of the EPM compared with control mice (p = 0.033). B, There was no effect of treatment on the percent of time spent in the open arms of the EPM when pooled data in A were separated according to sex. C, There was no effect of treatment on the number of open arm entries. D, There was no effect of treatment on the number of open arm entries when pooled data in C were separated by sex. E, There was no effect of treatment on the distance traveled during the EPM. F, Female mice traveled a significantly greater distance than male mice during the EPM test (p = 0.008). However, there was no effect of treatment in female and male cohorts. Error bars = SEM. *p <0.05.

Figure 10.

DREADD effects on hilar and GC c-Fos immunoreactivity. A1, A2, Representative c-Fos-immunoreactive (ir) cells in rostral and caudal coronal sections. Inset, scale bar, 20 µm. Control, eDREADD, and iDREADD mice were killed 90 min after completing HCNOE to evaluate c-Fos-ir cells. Mice were treated with CNO 90 min before HCNOE. B, All coronal sections of eDREADD mice had significantly more hilar c-Fos-ir cells than control and iDREADD mice (all p values < 0.001). C, When coronal sections were divided into rostral and caudal levels, both rostral and caudal sections had significantly more hilar c-Fos-ir cells per section in eDREADD mice compared with control and iDREADD mice (all p values < 0.001). D, All coronal sections of iDREADD mice had significantly more GCL c-Fos-ir cells per section than control and eDREADD mice (all p values < 0.001). E, When divided into rostral and caudal levels, both levels had significantly more GCL c-Fos-ir cells per section in iDREADD mice compared with control and eDREADD mice (all p values < 0.017). F1, F2, Representative c-Fos-ir in dorsal and ventral horizontal sections. Inset, Scale bar, 20 µm. G, All horizontal sections of eDREADD mice had significantly more hilar c-Fos-ir cells per section than iDREADD mice (p = 0.007). H, In dorsal horizontal sections, eDREADD mice had significantly more hilar c-Fos-ir cells per section than control and iDREADD mice (all p values < 0.028). There was no treatment difference in ventral sections. I, All horizontal sections of iDREADD mice had significantly more GCL c-Fos-ir cells per section than control and eDREADD mice (all p values < 0.007). J, In dorsal horizontal sections, iDREADD mice had significantly more GCL c-Fos-ir cells per section than control and iDREADD mice (all p values < 0.001). There was no treatment difference in ventral horizontal sections. Error bars = SEM. *p <0.05.

Figure 11.

DREADD effects on the MC circuit. A, eDREADD treatment increases MC firing and neurotransmitter release, which would facilitate both the (A1) direct MC→GC and (A2) indirect MC→GABAergic neuron→GC pathways. Notably, eDREADD treatment had a minimal effect on GCL c-Fos-ir, possibly because of opposing excitatory and inhibitory effects at the direct and indirect pathways, respectively. B, iDREADD treatment inhibits MC firing and neurotransmitter release, which would reduce MC effects at the (B1) direct MC→GC and (B2) indirect MC→GABAergic neuron→GC pathways. The reduced drive at the direct and indirect pathways appeared to promote GC firing, since iDREADD-treated mice showed significantly greater GCL c-Fos immunoreactivity. This finding is supported by previous studies that suggest that MC loss promotes GC excitability (Sloviter, 1991; Jinde et al., 2012; but see Ratzliff et al., 2004).