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- Maribel

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GABA-n inhibition :=)

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Presynaptic, but not postsynaptic, MORs resist acute desensitization

To examine the desensitization of presynaptic and postsynaptic MORs during exposure to ME, whole-cell voltage-clamp recordings were made from fluorescently labeled POMC neurons. Presynaptic and postsynaptic effects of ME were measured simultaneously. The MOR selective opioid agonist ME (30 μm) induced a postsynaptic outward current (42 ± 14 pA; n = 6; Fig. 1 A) that desensitized significantly within minutes of continued exposure ( p < 0.001, one-way repeated-measures ANOVA; τ = 116 s; plateau, 46% of baseline; Fig. 1 B). ME also caused a robust inhibition of the amplitude of eIPSCs (to 24 ± 3.5% of baseline; n = 14; p < 0.0001; Fig. 1 C, D). Inhibition of eIPSC amplitude was maintained throughout the superfusion of ME (30 μm; p = 0.27, one-way repeated-measures ANOVA; minutes 2 through 10; Fig. 1 C, D). Together, these results indicate that postsynaptic, but not presynaptic, MORs regulating POMC neurons acutely desensitize when exposed to a maximal concentration of ME similar to previous results with the agonist DAMGO ( Pennock and Hentges, 2011).

Presynaptic resistance to desensitization is a property of multiple Gi/o-coupled receptors

To determine whether differential presynaptic and postsynaptic desensitization may be a general property of G i/o-coupled receptors, or rather a specific property of MORs, the ability of GABA B, nociceptin, and κ-opioid receptors (KORs) to undergo or resist desensitization was examined. Superfusion of a maximal concentration of nociceptin (500 nm) induced a postsynaptic outward current (38 ± 4.8 pA peak) that declined within minutes to a plateau of 40% of baseline ( p < 0.0001, one-way repeated-measures ANOVA; τ = 210 s; n = 6; Fig. 2 A). A maximal concentration of nociceptin (500 nm) also caused an inhibition of eIPSC amplitude (to 30 ± 7.7% of baseline; n = 7; Fig. 2 B), but the inhibition of eIPSCs did not decline during the 10 min exposure to agonist ( p = 0.47, one-way repeated-measures ANOVA; minutes 2 through 10; Fig. 2 B, F). Similarly, the GABA BR agonist baclofen (30 μm, maximal concentration) induced an outward current (36 ± 6.0 pA; n = 6; Fig. 2 C) that declined during exposure (plateau, 53% of baseline; p < 0.0001, one-way repeated-measure ANOVA; τ = 84 s; Fig. 2 C). Baclofen also caused an inhibition of eIPSC amplitude (to 36 ± 3.4% of baseline; n = 19; Fig. 2 D) that was maintained throughout the exposure ( p = 0.053, one-way repeated-measures ANOVA; minutes 2 through 10; Fig. 2 D, F).

Activation of the KOR inhibits neurotransmitter release onto POMC neurons but has no apparent postsynaptic effects ( Pennock and Hentges, 2011). Superfusion of a maximal concentration of the KOR agonist U69593 (500 nm) resulted in inhibition of the amplitude of eIPSCs (to 66 ± 9.3% of baseline; n = 5; p < 0.0001; Fig. 2 E, F). Similar to the MOR, GABA BR, and nociceptin receptors, the KOR-mediated inhibition of eIPSC amplitude showed no desensitization during >10 min exposure to U69593 ( p = 0.071, one-way repeated-measures ANOVA; minutes 3 through 13; n = 5; Fig. 2 F). Nor-BNI (100 nm) was added upon the termination of U69593 superfusion to enhance wash/reversal ( Fig. 2 E). Together, these data show that resistance to acute desensitization may be a common characteristic of G i/o-coupled receptors located on presynaptic terminals.

Differential desensitization of presynaptic and postsynaptic Gi/o-coupled receptors in POMC neurons

It is plausible that the cell type on which a receptor is located, not the subcellular compartment in which it is found, determines whether or not that receptor will be resistant to desensitization. To determine whether receptors located in presynaptic and postsynaptic compartments of a single cell type show differential acute desensitization, experiments were performed in which desensitization of presynaptic receptors within POMC neurons was studied using an optogenetic approach. A viral vector containing a floxed ChR2 sequence was injected into the arcuate nucleus of mice expressing Cre recombinase under the control of the POMC promoter. In slices prepared from these mice, brief pulses of blue light depolarized the POMC neurons resulting in the release of neurotransmitter onto unidentified neighboring neurons from which recordings were made. Superfusion of ME (30 μm) resulted in a robust inhibition of light-evoked IPSC amplitude (to 36 ± 10% of baseline; n = 6; p < 0.0001; Fig. 3 A), which was reversed by the MOR antagonist CTAP (500 nm). Inhibition of the light-evoked IPSCs was maintained during the exposure to ME ( p = 0.14, one-way repeated-measures ANOVA; minutes 3 through 10; n = 6; Fig. 3 B), indicating that, unlike postsynaptic MORs in POMC neurons, MORs directly regulating transmitter release from POMC neurons do not undergo acute desensitization.

Baclofen (30 μm) also inhibited presynaptic release from POMC terminals as indicated by the reduction in light-evoked IPSC amplitude (to 27 ± 7.3% of baseline; n = 6; p < 0.0001; Fig. 3 C). This inhibition was maintained during continued agonist exposure ( p = 0.051, one-way repeated-measures ANOVA; minutes 2 through 10; n = 6; Fig. 3 D). Thus, both μ-opioid and GABA B receptors undergo desensitization in the postsynaptic ( Figs. 1, 2), but not presynaptic compartments ( Fig. 3 A-D).

Although KOR activation does not induce a GIRK-mediated current in POMC neurons ( Pennock and Hentges, 2011), the KOR agonist U69593 (500 nm) significantly inhibited the amplitude of light-evoked IPSCs (to 45 ± 14% of baseline; n = 4; p < 0.0001; Fig. 3 E). The inhibition of transmitter release was maintained during 14 min of continuous U69593 perfusion ( p = 0.94, one-way repeated-measures ANOVA; minutes 4 through 14; n = 4; Fig. 3 F). The inhibition of light-evoked IPSC amplitude was completely reversed by the KOR-selective antagonist nor-NBI (100 nm; Fig. 3 E). Together, the data from the light-evoked release studies demonstrate that the ability of some receptors to resist desensitization is likely dependent on the location of the receptor within the neuron.

Decreasing receptor number does not induce presynaptic desensitization

To determine whether receptor reserve may account for the lack of desensitization observed for MORs, the irreversible MOR antagonist β-CNA was used to reduce receptor reserve. β-CNA (50 nm) was superfused over the slice for 2 min following the washout of a brief application of ME (30 μm; Fig. 4 A, B). The pulse of ME before β-CNA application was used to determine the maximal inhibition possible for ME before receptor reserve was reduced. After the superfusion of β-CNA and a washout period (>10 min), ME (30 μm) was again applied. β-CNA reduced the inhibition of eIPSC amplitude caused by ME to 60 ± 11% of its original value (83 ± 3.4% inhibition of eIPSC amplitude before β-CNA vs 49 ± 8.0% inhibition of eIPSC amplitude after β-CNA; n = 3; p = 0.039, paired t test; Fig. 4 A, B). Although β-CNA treatment reduced the inhibition of eIPSC amplitude by ME, there was still no decrease in ME-mediated inhibition (desensitization) over the course of the drug application ( p = 0.065; n = 3; one-way repeated-measures ANOVA; minutes 2 through 11; Fig. 4 C).

To estimate the size of the MOR receptor reserve presynaptic to POMC neurons, dose-response curves were constructed before and after β-CNA treatment and analyzed using Furchgott's method ( Furchgott, 1966) ( Fig. 4 D, E). ME (30 μm) caused a much larger inhibition of eIPSC amplitude in control conditions compared with the inhibition after a 2 min exposure to β-CNA (79 ± 2.1% inhibition of eIPSC amplitude before β-CNA vs 42 ± 2.4% inhibition of eIPSC amplitude after β-CNA; p < 0.0001; n = 36, 12; Fig. 4 D). Additionally, there was an approximately threefold shift in the EC ₅₀ for ME after β-CNA (444 nm before β-CNA vs 1.336 μm after β-CNA). To estimate the fraction of the total number of presynaptic receptors inhibited by β-CNA, a double reciprocal plot of the agonist concentration needed to achieve a given inhibition of eIPSC amplitude before and after β-CNA treatment was constructed according to Furchgott's method ( Eq. 1). This analysis revealed that the 44% decrease in the maximal inhibition of eIPSC amplitude by ME corresponded to a 59-68% reduction in the total number of presynaptic MORs (95% confidence interval for q [0.3189, 0.4137]; Fig. 4 E) and provided an estimated K A value near that of the EC ₅₀ (95% confidence interval [0.693 μm, 1.315 μm]; Fig. 4 E) under control conditions. These data suggest that presynaptic resistance to desensitization is not due to receptor reserve since removing enough receptors to potently blunt the maximal response did not cause presynaptic receptors to display desensitization. Furthermore, the finding that removing ∼50% of the surface receptors reduces the functional response by ∼50% indicates that terminals regulating POMC neurons do not have a significant receptor reserve even under baseline conditions.

Morphine does not cause desensitization of MORs in the presynaptic compartment

MORs activated by morphine may desensitize through a mechanism distinct from those activated by ME or DAMGO in some systems ( Johnson et al., 2006; Kelly et al., 2008). Thus, it is possible that the environment of the presynaptic terminal may confer resistance to desensitization to a ME-activated MOR but not a morphine-activated receptor. To determine whether resistance to desensitization by presynaptic MORs may be agonist specific, eIPSC amplitude was measured during a 15 min superfusion of morphine (20 μm). Morphine caused a reduction in the amplitude of eIPSCs (to 52 ± 12% of baseline; n = 4; p < 0.0001) that was maintained throughout exposure to morphine ( p = 0.91; n = 4; one-way repeated-measures ANOVA; minutes 3 through 13; Fig. 5 A, B), indicating that presynaptic receptors do not undergo acute desensitization whether bound by full or partial agonists that likely confer different conformational states of the receptor.

Chronic morphine treatment (CMT) can reduce the efficacy of MOR agonists at presynaptic terminals ( North and Vitek, 1980; Fyfe et al., 2010) and enhance the extent and rate of acute desensitization of postsynaptic MORs ( Dang and Williams, 2005; Ingram et al., 2008). To determine whether CMT can modify presynaptic MOR signaling to enhance acute desensitization of these receptors in terminals presynaptic to POMC neurons, mice were treated with morphine (50 mg · kg −1 · d −1) or saline for 5-7 d using subcutaneous miniosmotic pumps before slice preparation. When slices were collected into morphine (1 μm) and maintained in morphine, application of naloxone (1 μm) caused a greater increase in the eIPSC amplitude (1.8 ± 0.13-fold increase in control; 4.4 ± 1.0-fold increase in CMT; n = 4-6; p = 0.01; Fig. 6 A, C) and decrease in the paired-pulse ratio in cells from CMT mice compared with saline-treated mice (0.68 ± 0.04 in control; 0.51 ± 0.05 in CMT; n = 4-6; p = 0.01; Fig. 6 A, B), indicating that morphine was effectively reaching the synapses and inhibiting transmitter release. Application of ME (30 μm) to slices from CMT mice that were collected and maintained in morphine-free aCSF resulted in a robust decrease in eIPSC amplitude (to 32 ± 3.2% of baseline; n = 20; p < 0.0001; Fig. 6 D, E) that was maintained over the course of an 11 min exposure ( p = 0.32; n = 5; one-way repeated-measures ANOVA; minutes 2 through 11) similar to that observed for tissue from untreated mice. Thus, presynaptic MORs resist desensitization after both acute and chronic exposure to morphine.

A fraction of presynaptic GABABRs acutely desensitize

It is plausible that a property of the presynaptic environment could somehow prevent receptor desensitization. However, in 7 of 26 recordings made from POMC neurons in which baclofen (30 μm) was applied while recording eIPSC amplitude, the inhibition of eIPSC amplitude declined during baclofen application ( p < 0.0001, one-way repeated-measures ANOVA; minutes 2 through 10; n = 7; Fig. 7 A, B). Inhibition peaked by the second minute of agonist exposure, on average, and desensitized by ∼50% by minute 10 (25 ± 6.9% of baseline at minute 2 vs 60 ± 7.4% of baseline at minute 10). In some recordings in which GABA B receptor desensitization was observed, it was possible to subsequently perfuse a maximal concentration of ME for >10 min. In these recordings, only the GABA B-mediated inhibition of eIPSC amplitude desensitized, whereas ME-mediated inhibition was maintained (data not shown). Additionally, GABA B-mediated inhibition of miniature IPSC (mIPSC) frequency was examined to determine whether ∼25% of all inputs onto POMC neurons contain GABA B receptors that are able to undergo desensitization, or whether only ∼25% of POMC neurons receive inputs with GABA B receptors that are able to undergo desensitization. Similar to what was observed when using eIPSCs to measure GABA B-mediated inhibition of neurotransmitter release, desensitization of GABA B-mediated inhibition of mIPSC frequency was only observed in 25% of recordings made (3 of 12 recordings; Fig. 7 C, D). This suggests that 25% of POMC neurons receive input from terminals on which GABA B receptors are able to undergo desensitization. If resistance or susceptibility of GPCRs to desensitization is due to a property of the terminals on which they are found, desensitization should have been observed in a similar fraction of recordings using agonists for the other receptors examined. Together, it appears that resistance to desensitization is likely conferred by a property of the receptor itself and not an intrinsic property of the terminals on which the receptors are found.

Differential presynaptic and postsynaptic desensitization of multiple Gi/o-coupled receptors

The ability of presynaptic, but not postsynaptic MORs to resist desensitization on POMC neurons is similar to the differential regulation of MORs previously reported in the periaqueductal gray (PAG) ( Fyfe et al., 2010) and the locus ceruleus ( Blanchet and Lüscher, 2002). Thus, sustained presynaptic MOR signaling is not unique to terminals within the arcuate nucleus. Differential desensitization of presynaptic and postsynaptic receptors also does not appear to be a phenomenon specific to the MOR since similar presynaptic resistance and postsynaptic susceptibility to desensitization was also found to occur for nociceptin and GABA B receptors regulating POMC neurons. κ-Opioid receptors, which were only found on presynaptic terminals, were also resistant to acute desensitization. Although KOR-induced postsynaptic currents were not observed in the present study, they likely possess the property of differential desensitization between presynaptic and postsynaptic receptors since KORs expressed in heterologous systems do display acute desensitization ( Henry et al., 1995). GABA BRs and adenosine A ₁ receptors (A ₁Rs) located in the presynaptic but not postsynaptic compartment of cultured hippocampal neurons also resist acute agonist-induced desensitization ( Wetherington and Lambert, 2002a, b), and a similar phenomenon has been described for GABA BRs regulating dopaminergic neurons in the VTA ( Cruz et al., 2004). Thus, the ability to maintain signaling may be a general property of a variety of presynaptic receptors located throughout the brain.

Presynaptic resistance to desensitization is independent of receptor reserve

The apparent lack of acute desensitization observed for presynaptic MORs could simply reflect that there is a high receptor reserve presynaptically such that there is always a sufficient pool of non-desensitized receptors to mediate maximal inhibition of transmitter release. This does not appear to be the case, however, since reducing MOR receptor reserve with β-CNA did not unmask any acute presynaptic MOR desensitization, although inhibition of transmitter release was significantly reduced. These results are consistent with a previous study examining presynaptic MORs in the PAG that also resist desensitization after knockdown with β-CNA ( Fyfe et al., 2010). The analysis of dose-response curves constructed under control conditions and after β-CNA exposure revealed that not only does resistance to desensitization by presynaptic MORs occur independent of receptor reserve, but that minimal MOR reserve is present on terminals presynaptic to POMC neurons to begin with.

CMT also can reduce receptor reserve ( Christie et al., 1987) and enhance both the extent ( Ingram et al., 2008) and rate ( Dang and Williams, 2005) of MOR desensitization following a subsequent acute application of agonist. However, in the present study, CMT did not induce desensitization of presynaptic MORs nor reduce the presynaptic inhibition induced by μ receptor agonists as has been observed in other studies ( North and Vitek, 1980; Fyfe et al., 2010). The presynaptic inhibition in the present study may be linked to the observation that terminals presynaptic to POMC neurons do not have a substantial receptor reserve even under basal conditions. Interestingly, GABA BRs and A ₁Rs located on the presynaptic terminals of hippocampal neurons also resist desensitization in a manner that is independent of receptor reserve ( Wetherington and Lambert, 2002a, b). These similarities across multiple cell types and multiple systems imply that not only is resistance to desensitization a common property of presynaptic G i/o-coupled receptors but that this resistance may occur through similar mechanisms. Furthermore, the fact that even prolonged exposure to agonist is not sufficient to reduce the presynaptic inhibition to a subsequent application of μ receptor agonist suggests that MORs in the presynaptic compartment resist desensitization by a specific mechanism rather than simply undergoing a postsynaptic-like desensitization in a slower manner.

Contribution of presynaptic environment in the resistance to acute receptor desensitization

MORs and GABA BRs undergo acute desensitization in the postsynaptic but not presynaptic compartment of POMC neurons as determined in the ChR2 experiments. This is consistent with previous studies showing that when hippocampal neurons form synapses onto themselves in culture, GABA B and adenosine A ₁ receptors in the somatodendritic compartment undergo acute receptor-specific (not effector-dependent) desensitization, but those receptors located in the presynaptic terminal do not desensitize ( Wetherington and Lambert, 2002a, b). Together, the data suggest that the cellular compartment in which a receptor resides may confer the tendency to undergo desensitization. However, the finding that presynaptic GABA BRs displayed acute desensitization in 10 of the 38 experiments (including both mIPSC and eIPSC experiments) in the present study indicates that compartmentalization alone may not be sufficient to confer resistance to desensitization. Furthermore, GABA BRs in CA3 neurons of the neonatal rat hippocampus located on the postsynaptic membrane or in presynaptic glutamatergic terminals do not desensitize with acute agonist exposure, although GABA BRs receptors located on GABAergic terminals desensitize readily ( Tosetti et al., 2004). Resistance to desensitization also occurs in postsynaptic GABA BRs located on GABAergic, but not dopaminergic, neurons of the VTA ( Cruz et al., 2004). These discrepancies between the patterns of presynaptic and postsynaptic desensitization of GABA BRs and the other receptors examined suggests that, although there is a strong correlation between compartmentalization and desensitization, compartmentalization alone is not sufficient to explain differential desensitization between presynaptic and postsynaptic G i/o-coupled receptors.

Such discrepancies between the GABA BR and the other receptors examined may be the result of variable expression of the GABA bR1 a/b splice variants. Although these splice variants are preferentially targeted to either terminal regions (R1 a) or somatodendritic regions (R1 b) of neurons, studies using R1 a and R1 b knock-outs suggest that either variant can be expressed at GABAergic terminals and postsynaptic membranes ( Vigot et al., 2006). Variable expression patterns of the GABA BR1 splice variants may explain the discrepancies between the patterns of presynaptic and postsynaptic desensitization of the GABA BR and other receptors that have been examined. Determining whether and why a certain GABA B R heterodimer composition is resistant to desensitization may provide insight into the mechanism by which other receptors resist acute desensitization.

Implications of differential presynaptic and postsynaptic receptor desensitization

The physiologic consequences of differential presynaptic and postsynaptic desensitization for specific receptors remain to be determined, but the data suggest that acute and chronic agonist exposure can differently affect cellular activity as postsynaptic responses may desensitize while presynaptic actions are maintained. In POMC neurons, for example, the initial action of MOR agonists is direct postsynaptic inhibition, whereas the prolonged effect of MOR activation would likely be disinhibition since the majority of inputs to POMC neurons are GABAergic ( Pinto et al., 2004; Hentges et al., 2009). Similar differential desensitization of GABA BRs located on GABAergic terminals and postsynaptic GABA BRs in the neonatal hippocampus has been suggested to underlie the induction of epileptiform discharges in this region ( Vardya et al., 2010). The lack of presynaptic MOR desensitization may explain the observation that synaptic transmission increases upon withdrawal of MOR agonist in multiple brain regions ( Bonci and Williams, 1997; Hack et al., 2003; Bie and Pan, 2005), consistent with a continued action at presynaptic MORs until removal of drug. This increase in synaptic transmission after the cessation of chronic opioid exposure has been implicated in withdrawal symptoms such as hyperalgesia ( Bie and Pan, 2005; Bie et al., 2005; Heinl et al., 2011). Thus, the differential presynaptic and postsynaptic desensitization of certain GPCRs may have significant functional consequences.

Conclusions

Whereas the postsynaptic actions of MORs and other G i/o-coupled receptors decline within minutes of agonist exposure, these same receptors on the presynaptic terminals of POMC neurons and presynaptic to POMC neurons continue to inhibit transmitter release. Thus, during continued agonist exposure, postsynaptic responses will likely decline while presynaptic actions will be maintained. The mechanisms limiting presynaptic receptor desensitization remain to be determined, but the data here suggest that compartment-specific modification of the receptor may underlie the resistance.

View Abstract

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Multiple Inhibitory G-Protein-Coupled Receptors Resist Acute Desensitization in the Presynaptic But Not Postsynaptic Compartments of Neurons

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Acute desensitization is a common property of Gi/o-coupled receptors. Recent data, however, suggest that, unlike μ-opioid receptors (MORs) located somatodendritically in neurons or expressed in heterologous systems, MORs in the presynaptic compartment of neurons are resistant to acute desensitization.

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Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA

Synaptic Structure Laboratory, Departamento de Ciencias Médicas, Facultad de Medicina, Instituto de Investigación en Discapacidades Neurológicas, Universidad Castilla-La Mancha, Albacete, Spain

Hippocampal interneurons comprise a diverse family of inhibitory neurons that are critical for detailed information processing. Along with gamma-aminobutyric acid (GABA), interneurons secrete a myriad of neuroactive substances via secretory vesicles but the molecular composition and regulatory mechanisms remain largely unknown. In this study, we have carried out an immunohistofluorescence analysis to describe the molecular content of vesicles in distinct populations of hippocampal neurons. Our results indicate that phogrin, an integral protein of secretory vesicles in neuroendocrine cells, is highly enriched in parvalbumin-positive interneurons. Consistently, immunoelectron microscopy revealed phogrin staining in axon terminals of symmetrical synapses establishing inhibitory contacts with cell bodies of CA1 pyramidal neurons. Furthermore, phogrin is highly expressed in CA3 and dentate gyrus (DG) interneurons which are both positive for PV and neuropeptide Y. Surprisingly, chromogranin B a canonical large dense core vesicle marker, is excluded from inhibitory cells in the hippocampus but highly expressed in excitatory CA3 pyramidal neurons and DG granule cells. Our results provide the first evidence of phogrin expression in hippocampal interneurons and suggest the existence of molecularly distinct populations of secretory vesicles in different types of inhibitory neurons.

Introduction

GABAergic neurons in the mammalian central nervous system (CNS) comprise a number of interneuronal subgroups that assemble into networks with specific synaptic properties and functional roles (reviewed in Freund and Buzsáki, 1996; McBain and Fisahn, 2001; Klausberger and Somogyi, 2008). A particularly vast heterogeneity of GABAergic cells has been reported in the hippocampal region where different subtypes can de distinguished by their distinct electrophysiological properties and specific neuropeptide content ( Freund and Buzsáki, 1996; McBain and Fisahn, 2001; Klausberger and Somogyi, 2008). Recent advances in understanding neuropeptide signaling suggest that the interneuronal system of neuropeptides is crucial for maintaining appropriate brain function. However, it is currently unknown how interneurons regulate neuropeptide storage and release to modulate synaptic transmission and information processing. This lack in our knowledge is in stark contrast to the detailed mechanistic insights into GABA exocytosis ( Südhof, 2013). Thus, a better understanding of the role of neuropeptide secretion in different interneuron types is necessary to unveil their function in regulating neuronal networks.

Neuropeptides and non-classical neurotransmitters are generally stored, delivered, and secreted via Large Dense Core Vesicles (LDCVs) which are described as large (70-150 nm) vesicles commonly found at non-specialized release sites ( Fried et al., 1985; Zhu et al., 1986). Additionally, neuropeptides can be found in medium sized vesicles (MDCVs; 60-100 nm) of 1/8 of the volume of LDCVs and predominantly located at presynaptic terminals ( van den Pol, 2012). Despite the remarkable variability of their cargo, LDCVs and MDCVs have been considered a molecularly homogenous population constituted by common integral proteins which can be found in both neuroendocrine and nerve cells. This is the case of the calcium-activator proteins for secretion (CAPS) and the chromogranin family ( Speidel et al., 2003; Machado et al., 2010; Bartolomucci et al., 2011). Chromogranin B (ChB, a.k.a. secretogranin I) expression has been reported in both neuroendocrine cells and neurons ( Fischer-Colbrie et al., 1985). Despite these commonalities in molecular composition, different modes of neuropeptide vesicle exocytosis have been reported in cortical and hippocampal neurons where vesicles with distinct cargo undergo either persistent or transient exocytosis ( de Wit et al., 2006; Farina et al., 2015).

Consistent with the notion of vesicle heterogeneity in the CNS, the present work has revealed that different hippocampal neurons exhibit distinct LDCV markers. Particularly, we have found that phogrin (a.k.a IA2β and PTPRN2) is highly expressed in parvalbumin positive interneurons (PV⁺). Phogrin is an integral protein of LDCVs in neuroendocrine tissues ( Wasmeier and Hutton, 1996) and has been implicated in several roles of the physiology of secretory vesicles including trafficking ( Wasmeier et al., 2002, 2005; Saito et al., 2011), exocytosis ( Cai et al., 2004), and endocytosis ( Torii et al., 2005; Wasmeier et al., 2005). To date phogrin has been widely used as one of the major auto-antigens in diabetes mellitus type I ( Kawasaki et al., 1996; Lu et al., 1996) and in the CNS, phogrin expression has been proposed to be developmentally regulated ( Chiang and Flanagan, 1996). Although the function of phogrin in nerve cells remains poorly understood, phogrin disruptions have been linked to attention deficits ( Lionel et al., 2011), addiction and mood disorders ( Yang et al., 2011), Down Syndrome ( Papoulidis et al., 2014) and HOXA1 spectrum disorder ( Abu-Amero et al., 2012) suggesting a pivotal role in regulating brain development and function.

Here, we have combined electron microscopy, immunohistofluorescence, and transgenic mice to reveal that phogrin is differentially expressed in different subtypes of hippocampal interneurons. Phogrin was found primarily expressed in hippocampal PV⁺ cells but virtually absent from somatostatin (SOM) and cholecystokinin (CCK) interneurons. In addition to being cell specific, phogrin expression was cargo-dependent, as it was also abundant in interneurons containing NPY, especially in the CA3-DG area. Our findings indicate the existence of different populations of secretory vesicles restricted to particular neuronal subtypes, and that phogrin may serve as a novel marker for specific interneuronal subgroups in the hippocampus.

Materials and Methods

Animals

Adult C5BL/6 wild-type mice were obtained from The Jackson Laboratory. PVCre-Ai6 mice were generated by breeding commercial PV-Cre mice (The Jackson Laboratory, J-008069) with reporter Ai6 mice that express the fluorescent protein ZsGreen1 upon Cre-mediated recombination (The Jackson Laboratory, J-007906). All animals used in this study were males. All animal procedures were performed in accordance of The University of Maryland School of Medicine Institutional Animal Care and Use Committee. The University of Maryland School of Medicine Institutional Animal Care and Use Committee approved the study. All efforts were made to minimize the number of animals used and their suffering throughout the experiments.

Immunohistofluorescence

Adult (P30-P60) C57BL/6 wild-type mice or PVCre-Ai6 mice were fatally anesthetized and transcardially perfused with ice-cold 4% paraformaldehyde in PBS. Brains were removed and post-fixed overnight (o/n) at 4°C in the same fixative solution. Coronal brain slices of 30 μm were obtained in a KD-400 vibrating microtome (IHC World), collected as floating sections and blocked for 1 h and a half at room temperature (RT) in a solution containing 0.3% Triton X-100 and 5% NGS in PBS. After blocking, sections were incubated (o/n; 4°C) with the following primary anti-bodies in a solution containing 0.3% Triton X-100 and 1% NGS in PBS: Rabbit anti-Phogrin (1:200; Torii et al., 2009); Mouse anti-Phogrin (3 μg/mL; Sigma-Aldrich SAB1406349); Mouse anti-VAMP2 (1:200; Synaptic Systems 103211); Mouse anti-GAD67 (1:1000; Sigma-Aldrich G5419); Rabbit anti-GAD67 (1.5 μg/mL; Boster PA1036); Rabbit anti-NeuN (1:500; Cell Signaling 12943); Rabbit anti-Chromogranin-B (1:200; Synaptic Systems 259103); Rabbit anti-Neuropeptide Y (1:2000; Peninsula laboratories T-4070); Rabbit anti-Somatostatin (1:1000; Peninsula laboratories T-4103); Rabbit anti-Cholecystokinin (1:2000; Sigma-Aldrich C2581). After five washes of 15 min each in PBS containing 0.25% Tween 20, the floating sections were incubated with specific Alexa conjugated antibodies (1:200, Molecular Probes) in a PBS solution containing 0.3% Triton X-100 and 1% NGS (for 2 h at RT. Finally, sections were washed, and incubated with DAPI (Sigma-Aldrich) for 10 min and mounted. Sections were stored at 4°C, and images were acquired on a Zeiss LSM510 Meta confocal scanning microscope.

Immunohistochemistry for Electron Microscopy

Electron microscopic examination of immunoreactivity for phogrin in the mouse dorsal hippocampus was performed as described previously using the pre-embedding immunogold method ( Luján et al., 1996). Briefly, free-floating sections were incubated in 10% NGS diluted in TBS for 1 h at RT. Sections were then incubated for 48 h in a Mouse anti-Phogrin antibody (Sigma-Aldrich SAB1406349) at a final protein concentration of 1-2 μg/ml diluted in TBS containing 1% NGS. After several washes in TBS, sections were incubated for 3 h in goat anti-mouse IgG coupled to 1.4 nm gold (Nanoprobes Inc., Stony Brook, NY) diluted 1:100 in TBS containing 1% NGS. After several washes in phosphate-buffered saline (PBS), the sections were postfixed in 1% glutaraldehyde diluted in the same buffer for 10 min. They were washed in double distilled water, followed by silver enhancement of the gold particles with a HQ Silver kit (Nanoprobes Inc., Stony Brook, NY). Then, sections were treated with osmium tetraoxide (1% in 0.1 M PB), block-stained with uranyl acetate, dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan (Fluka) resin. Regions of interest were cut at 70-90 nm on an ultramicrotome (Reichert Ultracut E, Leica, Austria) and collected on 200-mesh nickel grids. Staining was performed on drops of 1% aqueous uranyl acetate followed by Reynolds's lead citrate. Ultrastructural analyses were performed in a Jeol-1010 electron microscope.

Measurements and Quantification

All quantifications were obtained from a minimum of 10 sections from the dorsal hippocampus per mice. Immunofluorescence was analyzed in the different hippocampal subregions [CA1, CA2, CA3, and dentate gyrus (DG)] using approximately 20-30 fields of view for each condition. Both the granular cell layer and hilus were analyzed together and reported as the DG region. Colocalization of phogrin with NeuN, GAD-67 and interneuron markers was expressed as a percentage of number of positive cells out of phogrin positive cells. Colocalization with VAMP2 and Chromogranin B, and that of GAD67 with Chromogranin B, were expressed as Pearson's correlation coefficient ( Adler and Parmryd, 2010). To this end, the Intensity Correlation Analysis plugin in ImageJ (National Institutes of Health) was used ( Li et al., 2004), and rolling ball background subtraction was applied to the images before processing. Fields of view containing ventricles or dead pixels were digitally cropped for processing, since it has been reported that this background pixels could yield artifactual results ( Nakamura et al., 2007). Absolute percentages were estimated as the fraction of positive cells out of the total number of cells (identified by nuclear DAPI staining) in a given field of view. Normalized percentages were calculated as the absolute percentage of phogrin⁺ cells divided by the absolute percentage of GAD67⁺ cells for each hippocampal subregion. For VAMP2-phogrin and Chromogranin B-phogrin colocalization measurements, phogrin images were thresholded and a binary mask was obtained. This mask was subsequently used to analyze colocalization exclusively in those ROIs corresponding to phogrin positive puncta. When cell densities were expressed as a function of the overall number of cells, the Particle Analysis-Nucleus Counter plugin was employed over the DAPI images corresponding to each field of view; quantifications were then visually surveyed, to assure the proper functioning of this plugin.

Phogrin Antibody Validation

The commercially available antibody mouse anti-Phogrin (Sigma-Aldrich SAB1406349) primarily used in this study was validated against the home-made antibody rabbit anti-Phogrin generated by Torii et al. (2009). Double immunohistofluorescence using both antibodies yielded a Pearson's coefficient of 0.84 ± 0.01, indicating a high degree of colabeling of both antibodies in different hippocampal subregions (Supplemental Figures 1, 2) and several other brain regions (Supplemental Figure 3). Pearson's coefficient was calculated using the Intensity Correlation Analysis plugin as described previously in Measurements and quantification.

Statistics

Data were analyzed using OriginPro 8.0 software. For statistical purposes one-way ANOVA followed by Bonferroni's test was used. Differences were considered statistically significant when p < 0.05 with a confidence limit of 95%. All values were expressed as mean ± S.E.M.

Results

Interneurons Exhibit Different Vesicle Markers than Excitatory Cells in the Hippocampus

To identify the molecular composition of secretory vesicles in different hippocampal interneuron subtypes, we started by characterizing the expression pattern of canonical LDCV markers CAPS-1 and chromogranin B ( Speidel et al., 2003; Machado et al., 2010). According to previous work in primary cultured neurons ( Farina et al., 2015), immunohistofluorescence analysis in adult mouse slices revealed high levels of CAPS-1 (Supplemental Figure 4) supporting the notion that this protein is associated with neuronal LDCVs. As expected, CAPS-1 was broadly expressed in the hippocampus showing no particular cell-specificity. This was in contrast to chromogranin B staining which was abundant in neurons of the CA3 stratum pyramidale, stratum lucidum and in both the hilar region and the molecular layer of the DG (analyzed together as the whole DG region), but mostly absent from the CA1 and CA2 subregions (Figure 1A) consistently with previous reports ( Kroesen et al., 1996; Nicolay et al., 2007).

Figure 1. Endogenous Chromogranin B is not detected in GAD67+ Cells. (A) Distribution of endogenous Chromogranin B immunolabeling in the adult mouse hippocampus. Scale bar represents 100 μm. (B) Immunohistofluorescence analysis in CA3 and DG of the expression pattern of GAD67 and Chromogranin B showing a marked segregation in hippocampal tissue. Arrow heads indicate Chromogranin B −/GAD67⁺ cells. Left-bottom boxes show a magnified view of the area of interest delimited by the smaller box. Scale bar represents 40 μm. (C) Chromogranin B and GAD67 labeling and colocalization map obtained from ImageJ Intensity Correlation Analysis plugin. Scale bar represents 40 μm. (D) Mean Pearsons' correlation coefficient for GAD67 and Chromogranin B obtained from several CA3 and DG fields (Mean coefficient value = 0.12 ± 0.02; n = 48 fields).

Surprisingly, double labeling of chromogranin B and anti-GAD67 to identify GABAergic neurons ( Kanaani et al., 2010) yielded a low mean Pearson's coefficient value (0.12 ± 0.02; n = 48 fields; given the low expression levels of chromogranin B in the CA1 and CA2 regions, the mean coefficient value was obtained only from CA3 and DG regions) (Figures 1B-D). Our results indicate that both LDCV-associated proteins CAPS-1 and chromogranin B are not highly expressed in hippocampal interneurons. Particularly chromogranin B, which is primarily absent from GAD67 positive neurons, is unlikely to be an integral component of neuropeptide-containing vesicles in inhibitory cells. These results suggest that interneuronal LDCVs may contain different molecular constituents.

Phogrin: A Vesicle Marker of Hippocampal Interneurons

Given that commonly used LDCVs markers like CAPS-1 and chromogranin B cannot be used to definitely identify secretory vesicles in hippocampal interneurons, we reasoned that interneuronal vesicles may present a distinct molecular composition. We tested phogrin, a transmembrane protein of neuroendocrine vesicles which has been detected during early developmental stages of brain formations that give rise to inhibitory neurons ( Chiang and Flanagan, 1996). Because phogrin has been seldom probed in neuronal tissue, we analyzed phogrin expression using electron microscopy and immunohistofluorescence with two different antibodies. Commercially available Mouse anti-Phogrin (Sigma Aldrich SAB1406349) was tested against a Rabbit anti-Phogrin validated by Torii and colleagues for electron microscopy of pancreatic tissue ( Torii et al., 2009). As described in Materials and Methods, both antibodies showed nearly identical labeling patterns (Pearson's coefficient of 0.84 ± 0.01), indicating a high degree of co-labeling in the hippocampus (Supplemental Figures 1, 2) and several other brain regions (Supplemental Figure 3).

Immunohistofluorescence using a commercially available antibody, revealed that phogrin was expressed in discrete cells through all hippocampal regions analyzed (CA1, CA2, CA3 and DG). Phogrin labeling was observed in the stratum oriens, stratum radiatum and in the upper layer of stratum pyramidale (Supplemental Figure 2). Using double immunohistofluorescence against phogrin and the specific neuronal marker NeuN we confirmed that phogrin expression was limited to neurons in the different hippocampal subregions (colocalization percentages: CA1: 100 ± 0%; CA2: 100 ± 0%; CA3: 93.75 ± 4.58%; DG: 100 ± 0%; 91 NeuN⁺ cells out of 94 phogrin⁺ cells; Figures 2A,B). The morphology and localization of phogrin⁺ neurons suggested that phogrin may be primarily expressed in interneurons. To confirm this possibility we performed double immunohistofluorescence against phogrin and the GABAergic neuron marker GAD67. Surprisingly, the vast majority of phogrin-expressing neurons were GAD67 positive cells suggesting phogrin is a specific marker for interneuronal vesicles in the hippocampus (Normalized percentage as a fraction of GAD67⁺ cells: CA1: 92.54 ± 2.86%; CA2: 90.10 ± 5.34%; CA3: 90.20 ± 3.45%; DG: 77.38 ± 6.46%; 171 GAD67⁺ cells out of 197 phogrin⁺ cells; Figures 2C,D).

Figure 2. Phogrin is primarily expressed in interneurons. (A) Phogrin positive neurons express the pan neuronal marker NeuN. Scale bars represent 40 μm. (B) Quantification of NeuN expression in phogrin⁺ cells. Virtually all analyzed phogrin⁺ cells expressed NeuN (91 out of 94 cells). (C) Phogrin positive neurons co-express the interneuronal marker GAD67. Scale bars represent 40 μm. (D) Quantification of GAD67 expression in phogrin⁺ neurons. The vast majority of phogrin positive neurons were found to be GAD67⁺ (171 out of 197 cells) and no statistical differences were found between different hippocampal subregions. (E) Absolute percentages of GAD67 (black and gray bars) and phogrin (red bars) positive cells over the total number of cells measured as the total number of DAPI stained nuclei (624 GAD67⁺ cells and 248 phogrin⁺ cells out of 21,179 cells; * p < 0.05). (F) Percentage of phogrin⁺ cells normalized to the percentage of GAD67⁺ cells in each hippocampal subregion. One-way ANOVA followed by Bonferroni's test for means comparison; all the possible comparisons are non-significant. (G) Phogrin positive neurons lack Chromogranin B expression in CA3. Scale bar represents 40 μm. Chromogranin B expression is also absent in DG phogrin⁺ neurons. Scale bar represents 20 μm. (H) Colocalization map obtained after running Intensity Correlation Analysis Plugin in Image J. (I) Mean Pearsons' correlation coefficient for phogrin and Chromogranin B from several CA3 and DG fields (Mean coefficient values: Somas = 0.04 ± 0.04; Puncta = 0.05 ± 0.03; Two sample t-test for mean comparison, p = 0.87; Somas n = 7 fields; Puncta n = 11 fields). Boxes within A,C,G,H show a magnified view of the area of interest delimited by the smaller box.

Since most of phogrin⁺ neurons were also GAD67⁺ cells, we quantified the density of phogrin⁺ neurons as a fraction of the densities of GAD67⁺ cells. We found no differences between the densities of phogrin⁺ cells expressed as a percentage of GAD67⁺ cells throughout the different hippocampal regions (colocalization percentages: CA1: 42.40 ± 4.15%; CA2: 44.79 ± 5.76%; CA3; 55.58 ± 4.38%; DG: 49.96 ± 4.30%; Figures 2E,F). Our data indicate that contrary to previous suggestions ( Chiang and Flanagan, 1996), phogrin expression is abundant in the adult hippocampus and other brain regions (Supplemental Figures 2, 3). More importantly, phogrin is selectively expressed in inhibitory neurons which is suggestive of a specific role in the regulation of interneuronal vesicles in the postnatal hippocampus.

Phogrin does Not Colocalize with the Canonical LDCVs Marker Chromogranin B

Consistent with selective labeling of phogrin in hippocampal interneurons, we found a completely separation in the expression pattern of phogrin and chromogranin B in both puncta and somatic regions (Figures 2G-I) (Mean Pearsons' coefficient value: Somas = 0.04 ± 0.04; Puncta = 0.05 ± 0.03). Mean Pearsons' coefficient value was calculated in both CA3 and DG since chromogranin B was undetectable in CA1 and CA2 regions (Figure 1A). These results suggest that phogrin and chromogranin B are segregated in unique subpopulations of vesicles specific to inhibitory or excitatory hippocampal neurons, respectively.

Subcellular Expression of Phogrin in Hippocampal Interneurons

Given that the subcellular expression of phogrin in the postnatal brain has been seldom explored, we performed immunolabeling using a pre-embedding immunogold method in the CA1 region of the hippocampus (Figure 3A). Immunoparticles for phogrin were mainly located at presynaptic sites in axon terminals of basket cells establishing inhibitory synaptic contacts with the cell body of pyramidal cells. Furthermore, phogrin labeling was never detected in axon terminals establishing excitatory synapses recognized by the presence of a prominent postsynaptic density in the postsynaptic element. A significant proportion of immunoparticles for phogrin were also detected at postsynaptic sites in dendritic shafts of interneurons, as well as associated with the rough endoplasmic reticulum in the cell body of interneurons. We expanded our analysis by performing double immunofluorescence against phogrin and the synaptic vesicle protein VAMP2. VAMP2 (a.k.a synaptobrevin-2) is an integral component of the SNARE complex required to exocytose vesicles thus can be used to detect presynaptic localizations enriched with fast neurotransmitter-containing vesicles ( Südhof, 2013). Additionally, VAMP2 can also be part of postsynaptic membrane compartments where can mediate activity-dependent exocytic events at dendrites ( Jurado et al., 2013; Jurado, 2014). Both VAMP2 and phogrin showed a punctuated labeling typical of membrane associated-proteins (Figure 3B). Agreeing with previous findings in rat primary hippocampal neurons ( Jiang et al., 1998), VAMP2 and phogrin exhibited a high degree of colocalization in punctate regions but also in the somas (Figure 3C; Mean Pearsons' coefficient values: Somas = 0.70 ± 0.03; Puncta = 0.63 ± 0.02). The subcellular expression of phogrin-containing vesicles points to the possibility that these vesicles may coexist with small synaptic vesicles at presynaptic sites but also be stored at non-canonical sites like postsynaptic localizations.

Figure 3. Subcellular expression of phogrin in hippocampal interneurons. (A) Immunoreactivity for phogrin in the CA1 region of the hippocampus as revealed using a pre-embedding immunogold method. Immunoparticles for phogrin (black dots) were mainly located at presynaptic sites in button terminals (bt) opposing the cell body of pyramidal cells (arrow heads). Immunoparticles for phogrin were never detected in axon terminals of excitatory synapses recognized by the presence of a prominent postsynaptic density. A significant proportion of immunoparticles for phogrin (arrows) were also detected at postsynaptic sites in dendritic shafts (Den) of interneurons, as well as associated with the rough endoplasmic reticulum (large arrow heads). Cyt, cytoplasm; Nu, nucleus. Scale bars represent 0.5 μm. (B) Endogenous phogrin is co-expressed with VAMP2 in cell somas of stratum oriens and in puncta in stratum pyramidale in axo-somatic contacts. Scale Bar represents 5 μm. (C) Pearsons' correlation coefficients for VAMP2 and phogrin colocalization in both somas of stratum oriens and puncta in stratum pyramidale. (Mean coefficient values: Somas = 0.70 ± 0.03; Puncta = 0.63 ± 0.02. Two sample t-test for mean comparison, p = 0.18; Somas n = 4 fields; Puncta n = 14 fields).

Phogrin Is a Selective Marker of PV and NPY Interneurons

Given the prevalent expression of phogrin in GAD67⁺ neurons, we sought to identify which interneuron subtypes may selectively exhibit this newly characterized vesicle marker. Firstly, we carried out double staining between phogrin and CCK-containing interneurons (CCK), a subclass of basket cell interneurons which almost exclusively project to the stratum pyramidale ( Klausberger and Somogyi, 2008). Strikingly, CCK⁺ interneurons showed a total absence of phogrin in all hippocampal subregions analyzed (Figure 4; percentage of CCK⁺ cells out of phogrin⁺ cells: CA1: 0.00 ± 0.00%; CA2: 8.82 ± 6.41%; CA3: 4.32 ± 2.60%; DG: 0.00 ± 0.00%; 5 CCK⁺ cells out of 132 phogrin⁺ cells). Extended characterization to somatostatin positive (SOM⁺) interneurons, another prominent class of basket cells in the hippocampus, revealed almost identical phogrin exclusion (Figure 5; percentage of SOM⁺ cells out of phogrin⁺ cells: CA1: 7.14 ± 5.00%; CA2: 0.00 ± 0.00%; CA3: 7.50 ± 5.09%; DG: 0.00 ± 0.00%; 6 SOM⁺ cells out of 49 phogrin⁺ cells). These results indicate that phogrin expression is restricted to subpopulations of hippocampal interneurons.

Figure 4. Phogrin does not colocalize with CCK-positive interneurons in the hippocampus. (A) Phogrin is not co-expressed with CCK in CA1, CA2, CA3, or DG. Rigth-top boxes within each panel show a magnified view of the area of interest delimited by the smaller box. Scale bars represent 40 μm. (B) Colocalization percentage of phogrin⁺ and CCK⁺ interneurons throughout different hippocampal subregions (5 CCK⁺ cells out of 132 phogrin⁺ cells). Non-significant statistical differences were found between subregions. One-way ANOVA followed by Bonferroni's test for means comparison; all the possible comparisons are non-significant.

Figure 5. Phogrin does not Colocalize with SOM-positive Interneurons in the Hippocampus. (A) Phogrin is not co-expressed with SOM in CA1, CA2, CA3, or DG. Scale bars represent 40 μm. Rigth-top boxes within each panel show a magnified view of the area of interest delimited by the smaller box. (B) Colocalization percentage of phogrin⁺ and SOM⁺ interneurons throughout different hippocampal subregions (6 CCK⁺ cells out of 49 phogrin⁺ cells). Non-significant statistical differences were found between subregions. One-way ANOVA followed by Bonferroni's test for means comparison; all the possible comparisons are non-significant.

Since GAD67 expression is preferentially associated to PV⁺ interneurons ( Fukuda et al., 1997), we hypothesized that phogrin may be restricted to this interneuronal population. To better address this possibility, we performed single immunohistofluorescence against phogrin in PVCre-Ai6 mice to identify PV-containing neurons. Consistently, we found that most of the phogrin-expressing interneurons were PV⁺ in all the hippocampal regions examined (Figure 6; percentage of PV⁺ cells out of phogrin⁺ cells: CA1: 72.08 ± 6.17%; CA2: 79.79 ± 5.25%; CA3: 69.36 ± 7.24%; DG: 70.73 ± 5.56%; 225 PV⁺ cells out of 311 phogrin⁺ cells). Intriguingly, phogrin did not colocalize with PV⁺ interneurons in cortical areas (Supplemental Figure 5) suggesting that phogrin presence in these interneuronal subtype is region-specific. Lastly, we explored the colocalization of phogrin with NPY, another prominent peptidergic marker of hippocampal interneurons ( Freund and Buzsáki, 1996). We also observed strong colocalization between phogrin and NPY-expressing cells (Figure 7). Interestingly, the colocalization percentage of phogrin and NPY showed a gradual increase along different hippocampal subregions with the highest level of colocalization in the DG region (Figure 7; percentage of NPY⁺ cells out of phogrin⁺ cells: CA1: 34.91 ± 7.24%; CA2: 29.33 ± 9.43%; CA3: 54.16 ± 6.94%; DG: 80.83 ± 6.81%; 73 NPY⁺ cells out of 156 phogrin⁺ cells). Since we have found that phogrin expression is fairly homogenous throughout the different hippocampal subregions (Supplemental Figure 2), the increase in the colocalization ratio between phogrin and NPY may be likely due to higher levels of NPY expression in the DG ( Deller and Leranth, 1990; Gruber et al., 1994). Finally, we tested the percentage of NPY⁺ cells that also were PV⁺. As expected, the degree of colocalization was high (Figure 8A) and increased following the same pattern of phogrin and NPY co-expression (Figure 8B; Percentage of NPY⁺ cells out of PV⁺ cells: CA1: 14.93 ± 3.64%; CA2: 4.95 ± 1.70%; CA3: 39.75 ± 5.45%; DG: 62.78 ± 6.50%; 99 NPY⁺ cells out of 393 PV⁺ cells). Since PV⁺ and NPY⁺ interneurons are thought to belong to unique interneuronal populations, the addition of the colocalization percentages of PV⁺ cells / Phogrin⁺ cells and NPY⁺ cells / Phogrin⁺ cells was expected to be 100% maximum. Surprisingly, added percentages in CA3 and DG subregions were higher than 100% (Figure 8C) suggesting that a significant percentage of interneurons in these hippocampal regions contain both markers. Our findings indicate that phogrin is differentially expressed in a specific subgroup of basket cells which suggest a distinct molecular composition of neurosecretory vesicles in peptidergic hippocampal interneurons. Given its high specificity, phogrin can be used as a novel marker to identify PV-containing neurons in the whole hippocampus and NPY-containing PV⁺ cells in the CA3 and DG subregions.

Figure 6. Phogrin is highly expressed in PV-positive interneurons in the hippocampus. (A) Phogrin is co-expressed with PV in CA1, CA2, CA3, or DG. Arrow heads indicate phogrin −/PV⁺ interneurons. Rigth-top boxes within each panel show a magnified view of the area of interest delimited by the smaller box. Scale bars indicate 40 μm. (B) Colocalization percentage of phogrin⁺ and PV⁺ interneurons throughout different hippocampal subregions (225 PV⁺ cells out of 311 phogrin⁺ cells; n = 49 fields). Non-significant statistical differences were found between subregions. One-way ANOVA followed by Bonferroni's test for means comparison; all the possible comparisons are non-significant.

Figure 7. Phogrin and NPY show a region-dependent pattern of colocalization. (A) Phogrin is partially co-expresssed with NPY in CA1 and CA2 in contrast to the high degree of colocalization in CA3 and DG. Arrow heads indicate phogrin −/NPY⁺ interneurons. Rigth-bottom boxes within each panel show a magnified view of the area of interest delimited by the smaller box. Scale bars represent 40 μm. (B) Colocalization percentage of phogrin⁺ and NPY⁺ interneurons throughout different hippocampal subregions (73 NPY⁺ cells out of 156 phogrin⁺ cells; * p < 0.05). One way ANOVA followed by Bonferroni's test for means comparison; non-statistical significant comparisons are not indicated in the graph for clarity.

Figure 8. A specific subpopulation of PV+ interneurons also contains NPY. (A) NPY is partially co-expresssed with PV in CA3 and DG but not in CA1 and CA2 subregions. Arrow heads indicate PV −/NPY⁺ interneurons. Left-bottom boxes within each panel show a magnified view of the area of interest delimited by the smaller box. Scale bars represent 40 μm. (B) Colocalization percentages of NPY and PV in different hippocampal subregions (99 NPY⁺ cells out of 393 PV⁺ cells; * p < 0.05). (C) Bar graph represents overlapping mean colocalization percentages of phogrin⁺/NPY⁺ and PV⁺/NPY⁺ interneurons throughout different hippocampal subregions. Note that the value is greater than 100% in CA3 and DG. One way ANOVA followed by Bonferroni's test for means comparison; non-statistical significant comparisons are not indicated in the graph for clarity.

Discussion

Interneurons are often classified according to neuropeptide content which usually concurs with distinct electrophysiological properties (reviewed in Freund and Buzsáki, 1996; McBain and Fisahn, 2001; Klausberger and Somogyi, 2008). However, it is becoming increasingly clear that interneuronal neuropeptides are more than convenient neurochemical markers and can act as important modulators of neuronal activity ( Baraban and Tallent, 2004). In particular, interneuronal neuropeptides appear to play roles in cognition ( Dutar et al., 2002) and as endogenous anti-epileptic agents ( Erickson et al., 1996). Although neuropeptide release underlies the integrative role of interneurons, it is still unclear how specific neuropeptide content mediates interneuron function. In answering this question, it is important to elucidate the mechanisms of neuropeptide storage and secretion in distinct interneuronal subtypes.

Neuropeptides are confined to specialized vesicles (LDCVs and MDCVs) which are often released at non-canonical release sites in a slower manner than traditional neurotransmitters ( Fried et al., 1985; Zhu et al., 1986; van den Pol, 2012). Most of our knowledge of the molecular composition and exocytosis of secretory vesicles comes from studies in neuroendocrine tissue where a number of markers have been identified. Thus, the chromogranin family (a.k.a. secretogranins) and CAPS have been found in both endocrine and neuronal tissue ( Speidel et al., 2003; Machado et al., 2010; Bartolomucci et al., 2011). These findings pointed to the notion that despite the striking diversity of cargo, neuronal secretory vesicles may exhibit a fairly homogenous molecular composition. In contrast to this convention, our data demonstrates that different interneuronal types exhibit distinct vesicle markers (for a summary see Table 1).

Table 1. Summary of colocalization percentage and Pearson's correlation coefficient for different markers used in this study.

Using a combination of electron microscopy, immunohistofluorescence and transgenic mice, we have shown that chromogranin B, a canonical LDCV marker, is not expressed in GAD67⁺ neurons in the hippocampus (Figures 1B-D). Surprisingly, phogrin a receptor-type protein tyrosine phosphatase which has been extensively studied in endocrine cells ( Wasmeier and Hutton, 1996; Wasmeier et al., 2002, 2005; Torii et al., 2005, 2009) was found highly expressed in specific subtypes of hippocampal interneurons (Figures 4- 8). Although, phogrin in the mammalian CNS has been previously reported ( Chiang and Flanagan, 1996), its neuronal expression pattern and subcellular distribution have been only tangentially addressed. To the best of our knowledge this is the first report to identify phogrin expression in hippocampal interneurons. Furthermore, our findings clearly demonstrate that phogrin is highly expressed in PV⁺ interneurons throughout different hippocampal regions. These findings are consistent with the presence of phogrin mRNA in the primordial Medial Ganglionic Eminence (MGE) ( Chiang and Flanagan, 1996) that supplies the vast majority of PV⁺ hippocampal and cortical interneurons ( Tricoire et al., 2011). Intriguingly, our preliminary data suggest that phogrin colocalizes with PV⁺ cells exclusively in the hippocampus (Supplemental Figure 5). These results indicate that although their common origin, PV⁺ interneurons may exhibit different molecular content in a region-specific manner that may be related to distinct functionality. Furthermore, phogrin expression was tightly confined to a specific subset of interneurons since SOM⁺ and CCK⁺ cells, other prominent basket cell types in the hippocampus, lack phogrin expression altogether (Figures 4, 5). These results are in contrast with previous studies in neurosecretory cells of the gastrointestinal tract, in which phogrin can be found in both SOM⁺ and CCK⁺ cells ( Gomi et al., 2013) suggesting that phogrin expression is regulated in a tissue-specific manner.

Consistent with molecularly distinct pools of secretory vesicles, phogrin and chromogranin B, appeared markedly segregated in hippocampal neurons, with chromogranin B mainly confined to excitatory CA3 and DG neurons (Figures 1, 2). This segregated expression may not be exclusive to the mouse hippocampus as insulin-containing vesicles in pancreatic β-cells mostly contain phogrin with just a small fraction of vesicles with detectable chromogranin B ( Giordano et al., 2008). These data indicate that different neuronal populations display distinct LDCVs with non-overlapping molecular markers. In interpreting these results it is important to consider that while our immunohistofluorescence approach allows us to assess endogenous phogrin without the confounding of overexpression, it is limited by antibody specificity and sensitivity. In order to overcome these limitations, we have used two antibodies to confirm the expression of phogrin in hippocampal interneurons (Supplemental Figure 1). Antibody characterization confirms that both a commercially available antibody and an antibody generated for electron microscopy experiments by Torii et al. exhibit indistinguishable staining patterns (Supplemental Figure 1). Even so, we cannot rule out the presence of phogrin, although to a lesser extent, in excitatory neurons. However, the absence of chromogranin B in PV⁺ interneurons is strengthened by the undetectable levels of this protein in GAD67⁺ cells. Within inhibitory neurons, phogrin-containing vesicles exhibit a ubiquitous expression in both axon terminals and somato-dendritic compartments. Double immunohistofluorescence of VAMP2 and phogrin showed high colocalization levels in regions showing a punctate pattern of staining (Figures 3B,C) according to phogrin immunoparticles at axon terminals (Figure 3A). Additionally, a significant proportion of immunoparticles for phogrin were found at postsynaptic sites in dendritic shafts of interneurons suggesting that phogrin-containing vesicles may be stored at both presynaptic and postsynaptic regions. A postsynaptic location of phogrin is not surprising since somato-dendritic exocytosis has been described for many neuromodulators ( Fried et al., 1985; Zhu et al., 1986; van den Pol, 2012).

A pressing question is the function of phogrin in regulating vesicle dynamics in hippocampal interneurons. Previous studies in neuroendocrine cells and neurons proposed diverse roles of phogrin in LDCVs trafficking ( Wasmeier et al., 2002, 2005; Saito et al., 2011), exocytosis ( Cai et al., 2004), endocytosis ( Torii et al., 2005; Wasmeier et al., 2005), and cargo loading ( Henquin et al., 2008). Our data indicates phogrin may regulate distinctive functions depending on neuronal type and cargo content. Furthermore, taken together the observed cellular specificity of phogrin and chromogranin B challenges the classic view of secretory vesicles as molecularly homogenous vesicles. According to this, distinct modes of LDCVs exocytosis, transient and persistent, have been described in cortical and hippocampal neurons ( de Wit et al., 2009; Farina et al., 2015) implying the existence of different LDCVs pools in a similar fashion to small synaptic vesicles. In addition to the molecular composition, the balance between transient and persistent release may also be determined by LDCVs cargo ( de Wit et al., 2009).

Interestingly, we have found that a vast majority of phogrin⁺ cells in CA3 and DG neurons colocalized with NPY and that most of these neurons were also PV⁺ in a region-specific manner (Figures 7, 8). A potential explanation for the region specificity is that since both phogrin and PV expression is fairly homogenous throughout the different hippocampal subregions, the increase in the colocalization ratio may be likely reflecting the higher levels of NPY expression in the DG, which is well-characterized ( Deller and Leranth, 1990; Gruber et al., 1994). In this scenario, the higher ratio of colocalization in CA3 and DG neurons may just be reflecting the higher probability of NPY expression in these cells. Nonetheless, increasing numbers of NPY⁺ and PV⁺ interneurons is expected to have an impact in the regulation of excitatory synaptic transmission and information processing in these hippocampal subregions. Thus, NPY in the DG has been proposed to be an important factor in the modulation of epilepsy ( Erickson et al., 1996; Klapstein and Colmers, 1997; Gariboldi et al., 1998). Given the high levels of colocalization of phogrin with NPY, it would be interesting to address the potential implication of this protein in regulating NPY levels and its relationship to epileptic seizures. An interesting hypothesis would be that phogrin mediates NPY secretion in the hilar hippocampal region. In this scenario, phogrin loss of function may lead to aberrant hyperexcitability due to decreased levels of NPY. Therefore, cargo-specificity may be related with phogrin function in the adult hippocampus in the same manner as has been shown in neurosecretory cells ( Henquin et al., 2008). Undoubtedly, further experiments will be necessary to elucidate the role of phogrin in hippocampal neurons and secretory vesicle dynamics. Nonetheless, the identification of phogrin as a specific marker of vesicles in inhibitory cells in the mouse hippocampus will be a highly valuable new tool for the study of the role of interneuronal neuropeptide secretion in regulating neuronal networks.

Author Contributions

JR, FM, and SJ designed the work. JR performed the experiments and analyzed the data. RL performed electron microscopy experiments. JR, FM, and SJ wrote the manuscript.

Funding

NARSAD (Ref 22688) Young Investigator Award to SJ. Spanish Ministry of Education and Science (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to RL.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank Drs. Seiji Torii and Toshiyuki Takeuchi for their generous gift of the Rabbit anti-Phogrin antibody. We thank Dr. Joseph H. Mauben manager of the Confocal Microscopy Facility at the University of Maryland for his assistance.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fncel.2016.00202

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Excitatory and Inhibitory Neurons in the Hippocampus Exhibit Molecularly Distinct Large Dense Core Vesicles

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Hippocampal interneurons comprise a diverse family of inhibitory neurons that are critical for detailed information processing. Along with gamma-aminobutyric acid (GABA), interneurons secrete a myriad of neuroactive substances via secretory vesicles but the molecular composition and regulatory mechanisms remain largely unknown.

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Excel for EMG lab class data

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Two-pore channels (TPCs) are eukaryotic intracellular voltage-gated and ligand gated cation selective ion channels. There are two known paralogs in the human genome, TPC1s and TPC2s. In humans, TPC1s are sodium selective and TPC2s conduct sodium ions, calcium ions and possibly hydrogen ions. Plant TPC1s are non-selective channels. Expression of TPCs are found in both plant vacuoles and animal acidic organelles. These organelles consist of endosomes and lysosomes. TPCs are formed from two transmembrane non-equivalent tandem Shaker-like, pore-forming subunits, dimerized to form quasi- tetramers. Quasi-tetramers appear very similar to tetramers, but are not quite the same. Some key roles of TPCs include calcium dependent responses in muscle contraction(s), hormone secretion, fertilization, and differentiation. Disorders linked to TPCs include membrane trafficking, Parkinson's disease, Ebola, and fatty liver.

As implied by their name, TPC channels possess two pores and were named for their two Shaker-like repeats, which each have a pore domain. This contrasts with two-pore-domain potassium channels, which confusingly have only one pore and were named for the fact that each subunit has two P (pore) domains in its primary sequence.

Although much is left to be discovered about TPC function, they have been extensively studied thus far. Many questions have been raised about the specific function of TPC channels, as well as the ions and molecules that appear to be most closely affiliated with these channels. Some of these ions are sodium, calcium, and NAADP. Present knowledge of TPCs has come from experiments done on mice and plants, especially Arabidopsis thaliana. Additionally, because of the localization of these channels in mammals, it is difficult to use electrophysiological recordings on them. Therefore, these TPC channels have to be expressed in alternative compartments or organelles of the cell, such as plant vacuoles to be studied using the electrophysiological methods - especially the patch clamp technique. In order to clearly visualize the plant vacuoles, scientists have relied on fluorescent microscopy in their experiments. Using these techniques, scientists have been able to collect significant qualitative data in order to make conclusions about mammalian TPC functions. Specifically, scientists were able to conclude that human TPC are predominantly voltage-dependent sodium channels, and that PI(3,5)P2, an endolysosome-specific phosphoinositide (PIP), is a direct activator of TPC channels while NAADP is actually not an activator as it was once previously assumed to be.

At the mouth of the TPC pore, there are four amino acid residues with negative charges that can interact with ions that pass through. This site is too wide to select ions. Below the group of negative charges is the selectivity filter which is largely hydrophobic. There are two non-identical Shaker-like pore forming subunits. Subunit 1 consists of voltage sensing domain 1 (VSD1) and subunit 2 consists of the voltage sensing domain 2 (VSD2). The two subunit domains are separated by an EF-hand domain that has a calcium ion binding motif. This binding motif can facilitate channel activation by cytosolic calcium ions. Each of the two subunits are built from 12 transmembrane helices. The two central pore domains are combined together from the voltage sensing domains, VSD1 and VSD2. Both the N-terminal domain (NTD) and C-terminal domain (CTD) extend out on the cytosolic side, along with the EF-hand domain in the center that extends into the cytoplasm. The EF-hand domain extends into the cytosol, positioned between VSD1 and VSD2, where it can be activated by cytosolic calcium. The VSD2 domain is voltage sensitive active and can be inhibited by calcium in the lumen. This is a conformation change from the activation state to the inactive state. Two rings of hydrophobic residues seal the pore cavity from the cytoplasm; this results in forming the pore gate. Voltage sensors, selectivity filter, and the gate work together in a coordinated manner to open and close TPCs for regulation of ion conductance.

The VSD2 domain contains a normal voltage sensing motif, arginine residues R1, R2 and R3 and alpha helix S10, in respect to other voltage-gated ion channels structures, but this domain adopts a distinct conformation in the resting state of a voltage sensor. Luminal calcium acts as a TPC1 inhibitor, preventing ion conductance. There are two calcium binding sites for VSD2 on the luminal side. The first site does not affect the channel. Site 2, composed of residues in VSD2 and the pore domain, inhibits the channel by shifting the voltage dependence to more positive voltages.

Activation of TPCs is induced by a decrease in transmembrane potential, or by an increase in calcium concentrations in the cytosol. Low pH of the lumen and low calcium concentration could cause inhibition of these channels. TPCs are also phosphorylation-gated channels in both animals as well as plants. Sites of phosphorylation are at the N-terminal and C-terminal domains. These terminals are positioned to provide allosteric change in order to be activated by calcium from the cytosol.

Human and plant TPCs are multi-modal for conductance. The mechanism for channel opening is likely contributed to a combination of calcium concentrations, voltage, and phosphoregulation integration, in order to govern the conduction of ions through TPCs.

Biological roles (function/dysfunction)

Two-pore channels were analyzed by using cell biological methods, endolysosomal patch clamp techniques, and a variety of other methods to study their functions. From these, it was suggested that TPCs have some power in controlling the luminal pH in endolysosomal vesicles. When TPC2 expression is decreased or knocked out, there is a resultant elevation in production of melanin and thus melanosomal pH, and when TPC2 expression is increased, there is less production of melanin.

TPCs also are involved in nutrient detection as they become active constitutively on identifying the status of the nutrients. This is done by direct communication between the TPCs and mammalian/mechanistic targets of rapamycin (mTORs), which are associated with detecting levels of oxygen, nutrients, and energy in the cells and thus help with regulation of metabolism. This is how the TPCs play a role in this physiological regulation through this interaction.

TPCs regulate sodium and calcium ion conductance, intravasicular pH, and trafficking excitability. The second messenger nicotinic acid adenine dinucleotide phosphate ( NAADP) has been shown to mediate calcium release from these acidic organelles through TPCs. TPC2s are NAADP-gated calcium release channels where these TPC currents can be blocked by NAADP antagonists.

Various ailments can occur from the knockdown of these channels, from metabolic and general infectious diseases to even cancer. The pathological conditions due to this lacking of TPCs are covered in the following sections.

Membrane trafficking

TPCs play an integral role in membrane trafficking pathways. They are sectioned in endosomes and lysosomes, especially functioning in endo-lysosomal fusions. TPC trafficking activity has been noted to be conserved; but modifying TPCs affects transportation in the endocytotic pathway. The exact roles of TPCs are specific to cell type and context. These channels are permeable to calcium, making them function as Ca2+ ion channels. When stimulated by NAADP - a second messenger for TPCs -, calcium is released into the cytosol. The influx of calcium is what regulates the fusion between the endosome and lysosomes and what mediates trafficking events. When the function of TPCs are lost, substrates accumulate creating congestion. When the function of TPCs are increased, the lysosome becomes enlarged - which logically relates to increased fusion events with the endosome to lysosome.

Parkinson's disease

One implication of membrane trafficking dysfunction leads to Parkinson's disease. Mutations to LRRK2 enzyme alter autophagy dependent upon NAADP and TPC2. The mutation increases the amount of Ca2+ flow through TPC2 by NAADP evoked signals. This increase in signaling leads to an increase in size of the lysosomes due to the increased rate and amount of fusion. The lysosome, therefore, is not able to break down components the way it should. This inability is associated with the onset of the disease. As TPC2 plays a vital role in this specific mechanism of Parkinson's disease development, it may potentially be a therapeutic target.

The Ebolavirus takes advantage of host cell endocytotic membrane trafficking, leaving TPCs as a potential drug target. Ebolavirus enter cells through micropinocytosis with endosomal vesicles. After entrance into the endosomal vesicle, Ebolavirus membrane fuses with the endosomal membrane to release the viral contents into the cytosol before the endosome can fuse with the lysosome. For the movement of the virus in endosomes, Ca2+ is necessary. As NAADP regulates maturation of endosomes by the calcium release through TPCs, normal functioning of TPCs allows the Ebolavirus to escape. Therefore, when TPCs are not functioning, the Ebolavirus cannot escape before the fusion of the endosome with the lysosome. In fact, when mice are treated with tetradine the infection is inhibited. This is because tetradine blocks TPC functioning of calcium release and thus, the Ebolaviruses is contained within the endosomal network destined to be degraded by the lysosome.

Fatty liver

TPCs have been implicated in fatty liver diseases, such as NAFLD and NASH. As TPC2 is a cation channel for endocytotic membrane trafficking, TPCs contribute in trafficking LDL molecules for their breakdown and recycling. This primarily occurs within the liver. The degradation pathway causes LDL to end up in endosomes and lysosomes - where TPCs are located. The TPC mechanism once again allows the influx of calcium for the fusion of the endosomes and lysosomes (where LDL is degraded). When TPCs are not present, or are not functioning properly, the degradation pathway results in defected trafficking. Without the fusion event LDL accumulates in liver cells. The loss of TPCs have been found to be a cause of the yellow coloration of liver, an expression of fatty liver which indicates liver damage.

1. ^ Kintzer AF, Stroud RM (March 2016). "Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana". Nature. 531 (7593): 258-62. Bibcode:2016Natur.531..258K. doi:10.1038/nature17194. PMC . PMID 26961658. Other than Ca2+ and Na+ channels that are formed by four intramolecular repeats, together forming the tetrameric channel's pore, the new channel had just two Shaker-like repeats, each of which was equipped with one pore domain. Because of this unusual topology, this channel, present in animals as well as plants, was named Two Pore Channel1 (TPC1).

2. ^ Yu FH, Catterall WA (October 2004). "The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis". Science's STKE. 2004 (253): re15. doi:10.1126/stke.2532004re15. PMID 15467096.

3. ^ Patel S (July 2015). "Function and dysfunction of two-pore channels". Science Signaling. 8 (384): re7. doi:10.1126/scisignal.aab3314. PMID 26152696.

4. Grimm C, Holdt LM, Chen CC, Hassan S, Müller C, Jörs S, Cuny H, Kissing S, Schröder B, Butz E, Northoff B, Castonguay J, Luber CA, Moser M, Spahn S, Lüllmann-Rauch R, Fendel C, Klugbauer N, Griesbeck O, Haas A, Mann M, Bracher F, Teupser D, Saftig P, Biel M, Wahl-Schott C (August 2014). "High susceptibility to fatty liver disease in two-pore channel 2-deficient mice". Nature Communications. 5 (2): 4699. Bibcode:2014NatCo...5E4699G. CiteSeerX 10.1.1.659.8695. doi:10.1038/ncomms5699. PMID 25144390.

5. ^ Jammes F, Hu HC, Villiers F, Bouten R, Kwak JM (November 2011). "Calcium-permeable channels in plant cells". The FEBS Journal. 278 (22): 4262-76. doi:10.1111/j.1742-4658.2011.08369.x. PMID 21955583. The Arabidopsis two‐pore channel (AtTPC1) has been predicted to have 12 transmembrane helices and two pores (red lines).

6. ^ Robert Hooper (September 2011). Molecular characterisation of NAADP-gated two-pore channels (PDF) (Thesis). It is believed that TPCs, with their two pores, dimerise to form a functional channel.

7. ^ Lagostena L, Festa M, Pusch M, Carpaneto A (March 2017). "The human two-pore channel 1 is modulated by cytosolic and luminal calcium". Scientific Reports. 7: 43900. Bibcode:2017NatSR...743900L. doi:10.1038/srep43900. PMC . PMID 28252105.

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Two-pore channels (TPCs) are eukaryotic intracellular voltage-gated and ligand gated cation selective ion channels. There are two known paralogs in the human genome, TPC1s and TPC2s. In humans, TPC1s are sodium selective and TPC2s conduct sodium ions, calcium ions and possibly hydrogen ions. Plant TPC1s are non-selective channels.

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Can you find an actual image of a mitral cell (eg fluorescently  filled or stained)  

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Can you find an actual image of a mitral cell (eg fluorescently  filled or stained)  

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Spiny Stellate Neuron in the medial entorhinal cortex.

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by Micah and Sydney

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Shelby, Trent, Vicky