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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci Res. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2765820

GABA-mediated regulation of the activity-dependent olfactory bulb dopaminergic phenotype


Gamma-amino-butyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examined the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing GFP under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl-mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings showed that GABA promoted differentiation of SVZ-derived OB dopaminergic interneurons and suggested that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L- and P/Q-type Ca+2 channels as well as GABAA and GABAB receptors. These voltage-gated Ca+2 channels and GABA receptors have previously been shown to be required for the co-expressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the co-expressed GABAergic phenotype, and demonstrate a novel role for GABA in neurogenesis.

Keywords: GABA, dopamine, tyrosine hydroxylase, olfactory bulb, differentiation, slice culture


The glomerular and granule cell layer interneurons of the main olfactory bulb (OB) are derived primarily from the mammalian subventricular zone (SVZ), a proliferative region that generates neural progenitors and remains active throughout life (Alvarez-Buylla and Garcia-Verdugo 2002; Doetsch and Alvarez-Buylla 1996; Lois et al. 1996; Luskin 1993). Mature OB interneurons express a variety of neurotransmitter and neuroactive molecules, such as GABA and dopamine (DA) (Kosaka et al. 1998; Parrish-Aungst et al. 2007). The molecular mechanisms regulating the differentiation of OB DAergic interneurons, in particular, are not well-defined, but are clearly distinct from those in midbrain DAergic neurons (Cave and Baker 2008). OB DAergic interneurons are distributed primarily in the glomerular layer and can be readily identified by expression of tyrosine hydroxylase (Th), the first enzyme in the DA biosynthetic pathway. These interneurons receive excitatory glutamatergic input from both axo-dendritic synaptic contacts with olfactory receptor neurons (ORNs) and dendro-dendritic synapses with mitral and tufted (M/T) cells (Farbman 1992). Differentiation of OB DAergic neurons is activity-dependent since perturbations that prevent ORN synaptic activity produce a profound down regulation in DA production as well as the expression of both Th mRNA and protein (Baker et al. 1999; Baker et al. 1993). Studies with primary neonatal OB neuron cultures have shown that L-type Ca+2 channels are important for mediating the activity-dependent expression of TH (Cigola et al. 1998; McMillian et al. 1994; Puche and Shipley 1999). However, the mechanisms by which extrinsic signals and their associated membrane receptors in the OB glomerular layer provide positional information that initiates TH expression and terminal differentiation of OB DAergic neurons have not been elucidated.

GABA is expressed by a majority of OB interneurons, including the DAergic cells (De Marchis et al. 2007; De Marchis et al. 2004; Gutierrez-Mecinas et al. 2005; Kosaka et al. 1988; Kosaka et al. 1985; Parrish-Aungst et al. 2007; Puopolo and Belluzzi 1998). Release of both GABA and DA by OB periglomerular (PG) interneurons in response to excitatory input from either ORNs or M/T cells provides inhibitory feedback primarily through activation of D2 and GABAB receptors located on the ORN and M/T terminals in the glomeruli (Ennis et al. 2001; Keller et al. 1998). Release of GABA from PG interneurons can also inhibit neighboring PG neurons through dendro-dendritic synapses in the glomerular neuropil (Murphy et al. 2005; Smith and Jahr 2002).

In addition to its neurotransmitter role, GABA regulates the proliferation and migration rates of neuronal progenitors in the SVZ and RMS (Bolteus and Bordey 2004; Liu et al. 2005; Wang et al. 2003). In the OB granule cell layer, GABA also increases microtubule stability of interneuron progenitors which promotes the proper initiation, elongation and stabilization of dendrites (Gascon et al. 2006). Given these established developmental roles, a largely unexplored question is whether GABA can also modulate differentiation of specific interneuron phenotypes.

The present study reveals that GABA augments both Th promoter-driven GFP reporter gene (ThGFP) and TH protein expression in the glomerular layer of depolarized neonatal forebrain slice cultures. The findings suggest that differentiation of OB dopaminergic interneurons is regulated through a molecular pathway that is coupled to the co-expressed GABAergic phenotype.

Materials and Methods


ThGFP transgenic mice were obtained from Dr. Kazuto Kobayashi (Matsushita et al. 2002). Mice were housed in humidity-controlled cages at 22 °C under a 12:12 hour light:dark cycle and provided with food and water ad libitum. All procedures were carried out under protocols approved by the Cornell University Institutional Animal Care and Use Committee and conformed to NIH guidelines.

Slice Culture

Whole brains from mouse pups ranging in age from postnatal day 2 or 3 were dissected and placed into Petri dishes containing ice-cold dissection medium (Leibovitz’s L-15 media, pH 7.4, Life Technologies/Gibco BRL/Invitrogen, Carlsbad, CA). Brains were transected mid-sagittally and each hemisphere was placed with the medial face down into a small aluminum foil container and embedded in warm (37 oC) 3% low- melting-point agarose (Sigma, St. Louis, MO) in L-15 medium, then rapidly cooled on ice and cut into 200 μm sagittal slices with a vibratome (Model 1000; Ted Pella Inc., Redding, CA). Slices were cultured for 48–72 hours at 37°C and 5% CO2 in Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with B27 (1:50, Invitrogen), 0.5 mM L-glutamine (Invitrogen) and 25 μg/ml gentamycin (Sigma). Slices were either depolarized with 25 mM potassium chloride (KCl) or, as a control, treated with 25 mM sodium chloride (NaCl). The use of equimolar concentrations of either NaCl and KCl maintained a constant osmolarity and ionic strength of the slice culture media. The following pharmacological agents were used at the indicated final concentrations: GABA (Sigma) either 1, 10 or 100 μM; the L-type Ca+2 channel inhibitor, nifedipine (Sigma) at 10 μM; the P/Q inhibitor ω-agatoxin at 200 nM; the GABAA receptor antagonist bicuculline at 100 μM (Sigma); the GABAB antagonist CGP46381 at 100 μM (Tocris, Ellisville, MO); AMPA receptor antagonists, CNQX and DNQX at 20 μM and 10 μM, respectively (Sigma); the NMDA antagonist APV at 100 μM (Sigma) the mGluR antagonist, LY341495 at100 μM.

For focal stimulation, slices were cultured in an RC-26G recording chamber (Warner Instruments, Hamden, CT) with a circulating bath of oxygenated Neurobasal medium (Invitrogen) supplemented with B27 (1:50, Invitrogen), 0.5 mM L-glutamine (Invitrogen) and 25 μg/ml gentamycin (Sigma). The media was maintained at 37 °C with a in-line solution heater (Warner) that was controlled by TC-324B single channel controller (Warner). The olfactory receptor nerve layer of cultured slices were stimulated with a 2-contact CE2C75 cluster electrode (FHC Inc., Bowdoin, ME) using 1 second pulses at 100 Hz and 7 V applied at 10 minute intervals for 7 hours.


Localization of single antigens was performed as previously published (Saino-Saito et al. 2007). Briefly, sections were fixed for 30 min with phosphate-buffered (pH 7.2), 4% formaldehyde and washed in phosphate-buffered saline (PBS) before being blocked with 1% bovine serum albumin in PBS and incubated overnight with primary antisera. Antigens were visualized by incubation with appropriate biotinylated secondary antiserum, the Vector Elite kit (Vector Laboratories; Burlingame, CA) and 3,3′-diaminobenzidine (DAB, 0.05%) as chromogen with hydrogen peroxide (0.003%). Slides were dehydrated through a graded series of alcohols and cover-slipped. For double label immunofluorescence, secondary antibodies conjugated to either Alexa 488 or Alexa 594 were used (Molecular Probes/Invitrogen, Carlsbad, CA). Slides were cover-slipped with Cytoseal (VWR, Westchester, PA). Primary antibodies used were rabbit anti-TH (lot 15-2, raised in our laboratory, 1:25,000 and 1:10,000, for single and double-labeling, respectively), chicken anti-GFP (1:5,000 and 1,000; AB16901, Chemicon, Temecula, CA), mouse anti-TH monoclonal (1:10,000, for double labeling; Roche, Nutley, NJ) and goat anti-olfactory marker protein (OMP, kindly provided by Frank Margolis 1:7,000).

Quantitative Analysis

GFP fluorescence intensities were analyzed using the MetaMorph Imaging System (Molecular Devices, Sunnyvale, CA). Images of all slices were acquired with a Zeiss Axiomat microscope (Carl Zeiss, Thornwood, NY) equipped with a digital camera and KS400 software using the same filter sets and exposure times. Images were inverted and converted to 8 bit black and white in Adobe Photoshop (Adobe Systems Inc., San Jose, CA). The integrated pixel density of the glomerular layer was measured. To correct for differences in diameter, thickness and initial fluorescence intensity, the data for each slice was expressed as a ratio of the fluorescence intensity after 48 hours in culture to that at time zero.

Stereological cell counts were performed using confocal stacks of 20 images encompassing a total depth of 20 μm and a 0.212 mm2 area from equivalent sections from matched slices. A minimum of three regions in the sagittal sections (dorsal, anterior and ventral) from three slices were used for cell counts under each culture condition. All analyses were conducted using a Zeiss Meta 510 scanning confocal microscope and Zeiss LSM Image Browser software.

For all quantitative analyses results are expressed as a mean ± standard deviation. Statistical significance was analyzed by two way ANOVA with appropriate post-hoc tests using StatView Software (Statview Software Inc, Cary, NC). Results were considered significant if p<0.05.


Activity dependent OB ThGFP expression in the forebrain slice culture model system

To investigate the effect of GABA on activity-dependent OB TH expression, organotypic forebrain slice cultures were prepared from ThGFP transgenic mice containing a GFP reporter gene driven by the 9kb upstream Th gene promoter. Previous studies have demonstrated that this Th regulatory region is sufficient to mediate synaptic-activity dependent gene expression in the OB (Min et al. 1996; Saino-Saito et al. 2004). Although immunohistochemistry is sufficient both for labeling TH-expressing cells in tissue and observing qualitative changes in expression levels, this method cannot be used to quantify differences in TH expression levels between slices cultured under different conditions. By contrast, the ThGFP transgenic mice allow for quantitative analysis of Th promoter activity by fluorescence intensity measurements. In this study, ThGFP fluorescence intensities in living slices were measured at 0 and 48 hours in culture, and calculation of the fluorescence intensity ratio (48 hours relative to 0 hours) corrected for differences in diameter, thickness and initial fluorescence intensity of individual slices.

Addition of depolarizing concentrations of KCl to the culture media were used to simulate the afferent synaptic input necessary for induction of OB TH expression. The elevated KCl concentration (25mM) in this study is commonly used to induce neuronal depolarization in both primary and organotypic slice cultures (Cirrito et al. 2005; Li et al. 2001; Lohmann et al. 1998; Shahar et al. 2004; Shalizi et al. 2006; Suzuki et al. 2005). In KCl-depolarized forebrain slice cultures, there was almost total overlap in ThGFP and TH protein expression in the glomerular layer, whereas only ThGFP was expressed in the superficial granule and mitral cell layers (Fig. 1B). These expression patterns were nearly identical to those observed in vivo (cf. Fig. 1B vs. Fig. 1C) (Akiba et al. 2007; Saino-Saito et al. 2004).

Figure 1
Laminar expression pattern of TH and ThGFP in the OB. A, diagram of OB laminar organization: olfactory nerve layer (light gray), glomerular layer (red), glomeruli (yellow), external plexiform layer (dark green), mitral cell layer (light blue), internal ...

TH expression in OB DAergic neurons requires glutamatergic input from either axo-dendritic synapses with olfactory receptor neurons (ORN) or dendro-dendritic synapses with M/T cells (Fig. 2A). Although the M/T dendritic projections remain largely intact during slice culture preparation, the ORN axonal projections are severed from their respective soma. Despite being severed, focal stimulation of the ORN axons induced ThGFP expression and showed that the OB microcircuits necessary for activity-dependent induction of OB TH expression are preserved in the slice culture model system (Fig. 2B and 2C). To confirm that glutamatergic pathways modulated ThGFP expression in the slice culture model system, KCl-depolarized slices were treated with selective glutamate receptor antagonists (Fig. 2D). In these experiments, a similar and significant reduction in ThGFP expression was observed following either individual or combined treatment with antagonists for AMPA receptors, NMDA receptors and metabotropic glutamate receptors (DNQX, APV and LY341495, respectively). Consistent with a role for activation of excitatory microcircuits, ThGFP expression was also significantly reduced in depolarized slice cultures treated with tetrodotoxin (TTX), an inhibitor of TTX-sensitive Na+ channels (Fig. 2E).

Figure 2
Induction of ThGFP expression in forebrain slice cultures. A, diagram of the synaptic connections in a typical glomerulus. Olfactory receptor neuron axon terminals (black) form axo-dendro-dendritic synapses with mitral/tufted projection neurons (gray) ...

Synaptic activity-dependent gene expression is mediated by Ca+2 influx and activation of second messenger signal transduction pathways in several neuronal systems (Chawla 2002; West et al. 2001). Previous studies have also shown that L-type Ca+2 channels are necessary for depolarization-induced TH expression in neonatal primary OB neuron cultures (Cigola et al. 1998; McMillian et al. 1994; Puche and Shipley 1999). Consistent with these previous findings, ThGFP expression was reduced to basal control levels in depolarized slice cultures treated with nifedipine, an L-type Ca+2 channel blocker (Fig. 2F). These findings indicated that induction of ThGFP expression in depolarized slice cultures was dependent on L-type Ca+2 channels.

GABA enhances activity-dependent ThGFP expression in the glomerular layer

Previous reports have shown that GABA can depolarize OB glomerular interneurons (Murphy et al. 2005; Smith and Jahr 2002). However, GABA was not sufficient to induce ThGFP expression by itself in the slice cultures (Fig. 3A). By contrast, GABA enhanced ThGFP expression levels in a dose-dependent manner when the slice cultures were simultaneously depolarized with KCl (Fig. 3A, 3B and 3D–H). The addition of 10 μM GABA was the lowest concentration in these dose-response studies that generated a significant increase in ThGFP expression levels relative to depolarized slices not treated with GABA. For this reason, 10μM GABA was used for all subsequent experiments involving the addition of GABA. Also, GABA did not induce transgene expression in depolarized slices co-treated with either the L-type Ca+2 channel blocker nifedipine (Fig. 3C) or glutamate receptor antagonists (Fig. 3I). This finding was consistent with synaptic activity being prerequisite for GABA to enhance ThGFP expression levels.

Figure 3
GABA increases ThGFP expression in depolarized slice cultures. A, GABA increases ThGFP expression in slice cultures that are simultaneously depolarized with KCl, but GABA does not induce ThGFP expression in control NaCl-treated slices. B, GABA produces ...

GABAA and GABAB receptors are co-expressed with TH in the OB (Bonino et al. 1999; Panzanelli et al. 2005). To identify the specific receptors necessary for the GABA-mediated increase in ThGFP expression, KCl-depolarized slice cultures were treated with GABA and either the GABAA or GABAB receptor antagonists, bicuculline and CGP46381, respectively. Application of either antagonist blocked the enhancement of ThGFP expression induced by GABA, but neither antagonist had an effect on transgene expression in the absence of exogenous GABA (Fig. 3J). Prolonged excitation (≥15 minutes) of OB PG interneurons has been reported to produce a gradual rundown in the ability of these cells to release GABA (Smith and Jahr 2002). Since the slice cultures in this study were examined 48 hours after depolarization, the endogenous GABA levels were likely insufficient to generate a measurable effect on ThGFP expression and, therefore, an exogenous source of GABA was required to modify transgene expression.

The GABA-mediated enhancement in ThGFP expression in the glomerular layer could be produced by either an increase in the number of cells expressing ThGFP or by an increase in the transgene expression levels per cell. In equivalent regions of matched slices, stereological cell counts revealed that depolarization conditions in the absence of GABA did not significantly change the total number of GFP+ cells (Fig. 4A, 4B and and5A).5A). By contrast, the fluorescence intensity per GFP+ cell substantially increased under these conditions, notably in the processes of the PG cells (Fig. 4B). Together, these results indicated that the increase in GFP fluorescence induced by KCl-mediated depolarization resulted from an up-regulation of GFP expression in cells with already detectable levels of GFP at the time of slice culture preparation.

Figure 4
GABA increases co-expression of TH and ThGFP in depolarized slice cultures. Expression of ThGFP (green, A – C) and TH (red, D – F) in slices treated with NaCl, KCl and KCl with GABA. The merged images are shown in G – I. KCl increases ...
Figure 5
The number of GFP+ and TH+ cells relative to fluorescence intensity per GFP+ cell in slices cultures. A, number of GFP+ cells in equivalent regions of matched slices treated with either NaCl, KCl, or KCl with GABA. B, relative fluorescence intensity per ...

An equivalent analysis of depolarized slice cultures treated with GABA, revealed that the total number of GFP+ cells increased relative to cultures treated with only KCl (Fig. 4B, 4C) without changing the GFP fluorescence intensity per GFP+ cell (Fig. 5B). These findings revealed that the GABA-mediated enhancement of GFP fluorescence in the glomerular layer resulted from an increased number of cells expressing the transgene. Stereological cell counts performed on the same sections used for the ThGFP analysis showed that GABA also increased the number of PG cells expressing the endogenous TH protein (Fig. 4D–F and Fig. 5C).

In contrast to the lack of change in the number of ThGFP cells, KCl-mediated depolarization in the absence of GABA significantly increased the number of TH+ cells relative to NaCl controls (Fig. 4D, 4E and Fig. 5C). These differences may be attributed to the detection sensitivity of the GFP protein as compared to the endogenous TH protein. Under the non-depolarizing conditions, the low KCl concentration (5 mM) inherent to the culture media was likely sufficient to induce weak levels of Th promoter activity. However, there are an estimated 20 copies of the ThGFP transgene in the diploid transgenic mouse genome, as compared to the endogenous 2 copies of Th (Matsushita et al. 2002). This large difference in copy number likely results in greater expression levels of the GFP protein relative to the endogenous TH protein, which results in a greater detection sensitivity for the GFP protein.

P/Q-type Ca+2 channels are necessary for GABA to enhance ThGFP expression

Strong activation of L-type Ca+2 channels has previously been reported to stimulate opening of P/Q-type Ca+2 channels in OB interneurons (Murphy et al. 2005). To ascertain if P/Q type channels were also involved in the L-type Ca+2 channel-mediated induction of ThGFP expression, depolarized slice cultures were treated with ω–agatoxin, a P/Q-type Ca+2 channel-specific antagonist. In these studies, ω–agatoxin specifically blocked the GABA-mediated increase of ThGFP expression without diminishing the KCl-induced response (Fig. 6). Since activation of P/Q Ca+2 channels is critical for triggering dendritic release of GABA in OB PG interneurons (Murphy et al. 2005), these observations indicated that both GABA release and the enhancement of TH expression by GABA in glomerular interneurons were dependent on activation of both L- and P/Q-type Ca+2 channels in response to excitatory input.

Figure 6
The P/Q-type Ca+2 channel blocker, ω-agatoxin, prevents the GABA-mediated increase of ThGFP expression in depolarized slice cultures without diminishing the ThGFP expression levels induced by depolarization. Single asterisk indicates values that ...


Modulation of OB dopaminergic interneuron differentiation by GABA

In the current study, GABA induced ThGFP expression in a subset of depolarized glomerular layer cells through mechanisms that required L- and P/Q-type Ca+2 channels as well as GABAA and GABAB receptors. This GABA-mediated induction of ThGFP expression required depolarization as GABA, alone, was not sufficient. These results indicated that the number of depolarized OB glomerular cells expressing ThGFP was proportional to the external GABA concentration. Consistent with this finding, the increase in ThGFP expression levels mediated by GABA was dose-dependent. GABA also induced TH protein expression in the ThGFP-containing glomerular interneurons indicating that these cells were likely dopaminergic. Since the presence of TH is considered a marker of terminal differentiation in OB DAergic neurons, these findings suggest that GABA modulates maturation of the OB DAergic phenotype.

OB interneurons, including the glomerular DAergic cells, are generated throughout life, which results in a continuous ensemble of undifferentiated precursors among the differentiated neurons in the OB. The finding that GABA did not increase ThGFP levels per cell suggests that GABA does not directly modulate TH expression in differentiated OB DAergic neurons. Rather, the data support a model in which GABA indirectly regulates TH expression in OB DAergic neurons by modulating the maturation rate and formation of synaptic connections necessary for TH expression (Fig. 7). Consistent with this interpretation, previous studies have shown that GABA is crucial for synaptogenesis and integration of neuronal progenitors into developing neural circuits (reviewed in Akerman and Cline 2007; Ge et al. 2007). In the OB granule cell layer, GABA modulates maturation of interneuron progenitors by regulating the initiation, elongation and stabilization of dendrites (Gascon et al. 2006). In the present context, GABA may also promote both the development of dendritic projections and establishment of synaptic connections in OB DAergic progenitor cells, which enables the progenitors to receive the synaptic input necessary to induce TH expression (Fig. 7). Once synaptic connections are formed and glutamatergic input is received, TH expression is induced and differentiation is completed. Although the results of the GABA titration experiments indicate that the maturation rate is proportional to the ambient levels of GABA, PG interneurons in vivo typically require approximately four weeks to fully develop their dendritic and axonal morphologies (Belluzzi et al. 2003). Consistent with the model proposed here, these previous studies also indicated that GABAergic input precedes glutamatergic input in the maturation process of these interneurons (Belluzzi et al. 2003; Carleton et al. 2003).

Figure 7
Mechanistic model for differentiation of OB DAergic neurons. In the glomerular layer, undifferentiated OB DAergic precursors are depolarized by ambient GABA. This depolarization sequentially activates L- and P/Q-type Ca+2 channels which stimulates GABA ...

An alternative mechanism by which GABA could mediate an increase in the number of ThGFP and TH expressing cells in the glomerular layer is through an increase in either the proliferation or migration rates of OB DAergic progenitors. However, this seems unlikely since the concentration of GABA (10 μM) used in the studies to induce TH expression substantially reduced progenitor migration rates in the RMS (Bolteus and Bordey 2004). In addition, the depolarization conditions used in the current study (25 mM KCl) also increase the ambient levels of GABA in the RMS, which would further reduce progenitor migration (Bolteus and Bordey 2004). Similarly, GABA decreases progenitor proliferation rates in the RMS and SVZ (Liu et al. 2005), which are already low (Lois and Alvarez-Buylla 1994).

A striking finding in this study was the molecular inter-relationship between the co-expressed OB GABAergic and DAergic phenotypes. Although the expression of the GABA synthesizing enzymes (GAD 1/2) in the OB are not activity-dependent (Baker et al. 1988), the release of GABA from PG interneurons is activity-dependent and requires L- and P/Q-type Ca+2 channels (Murphy et al. 2005). Our results indicate that both of these Ca+2 channels are also required for the GABA-mediated induction of TH expression, suggesting that these Ca+2 channels are also required for the maturation of OB DAergic precursor cells. The current studies also indicated that L-type channels remain critical for TH expression in differentiated OB DAergic neurons. Thus, the co-expressed DAergic and GABAergic phenotypes are coupled by a common subset of voltage gated Ca+2 channels.

Although GABA can depolarize and activate L-type channels in OB DAergic precursors cells, the current study showed that GABA by itself is not sufficient to induce TH expression. This unexpected finding suggests that L-type Ca+2 channels are necessary, but not sufficient for activity-dependent reduction of TH expression. Thus, additional membrane receptors/channels and their cognate signaling pathways activated by either KCl-mediated depolarization or glutamatergic input are also necessary for TH expression in OB DAergic neurons. Consistent with this possibility, preliminary slice culture studies indicate that the activity-dependent synaptic release of the neurotrophin, BDNF, can also enhance depolarization-induced ThGFP expression (unpublished observation; YA, HB and JWC).

Activity-dependent TH expression in a forebrain slice culture model system

The current study demonstrated that KCl-depolarized slice cultures are an effective model system to study activity-dependent TH expression in the OB. Although bath application of KCl broadly depolarizes neurons, the ability of inhibitors for glutamate receptors and TTX-sensitive channels to reduce ThGFP expression levels in depolarized slices indicated that several key components of physiologically-relevant microcircuits necessary for induction of TH expression were functional in this model system. Some variability in the induced levels of ThGFP expression relative to non-depolarized (NaCl-treated) slices was also observed, but this likely resulted from fluctuations in basal ThGFP expression levels. Since the ThGFP expression levels were expressed as a ratio of fluorescence intensity at 48 hours relative to 0 hours, variation in basal expression levels measured at 0 hours affect the magnitude of the relative ratio. Despite this variability, the qualitative response to KCl and GABA were consistent throughout the study.

The current slice culture studies also show that L-type Ca+2 channels are essential for activity-dependent TH expression. These findings are consistent with both primary culture studies that suggested the importance of L-type channels (Cigola et al. 1998; McMillian et al. 1994; Puche and Shipley 1999) and electrophysiology studies that identified L-type channels as the major carrier of Ca+2 current in OB DAergic neurons (Pignatelli et al. 2005). In other neuronal systems, L-type Ca+2 channels modulate gene expression through activation of transcription factors, including CREB and AP-1 (Chawla 2002; West et al. 2001). The TH gene proximal promoter contains evolutionarily conserved binding sites for CREB and AP-1, both of which are necessary for TH expression in the OB (Baker et al. 2001; Liu et al. 1999; Trocme et al. 1998). Together, these data suggest that odor mediated activation of L-type Ca+2 channels induces TH expression in DAergic PG cells through the activity of CREB, AP-1 and related transcription factors.

Consistent with the critical role of L-type Ca+2 channels for the induction of TH expression, the L-type channel blocker, nifedipine, reduced ThGFP expression to basal levels. The residual ThGFP expression following treatment with either TTX or glutamate receptor antagonists was likely produced by direct KCl-mediated activation of L-type Ca+2 channels in the OB DAergic neurons. The CNS expresses two major subclasses of L-type channels, CaV1.2 and CaV1.3, that are distinguished by the presence or absence of α1C and α1D subunits, respectively (Lipscombe et al. 2004). Both subclasses are expressed in the OB glomerular layer (Ludwig et al. 1997; Tanaka et al. 1995). Although L-type channels are generally activated at high voltages, there are significant functional differences between the subclasses, including activation voltage thresholds (Lipscombe et al. 2004). Previous reports have shown that activation of CaV1.3 channels occurs at potentials as low as −55 mV, with half activation potentials of −40 mV (Xu and Lipscombe 2001). These voltages are approximately 20–25 mV more hyperpolarized than corresponding potentials observed for CaV1.2 channels under the same conditions. This lower activation threshold of CaV1.3 channels is likely to be significant since Ca+2 channels in OB DAergic neurons have been reported to activate at membrane potentials between −50 and −60 mV (Pignatelli et al. 2005; Puopolo et al. 2005). Under the 25 mM KCl-mediated depolarizing conditions used in the current study, Goldman-Hodgkin-Katz calculations predict a neuronal membrane potential of approximately −47 mV (see Supplemental Fig. 3), which is sufficient to partially activate CaV1.3 L-type Ca+2 channels.


The studies reported here indicate that GABA can modulate differentiation of OB DAergic neurons. This novel finding suggests that the regulation of dendritic outgrowth and stability as well as synaptogenesis by GABA also controls the number of OB glomerular interneurons expressing activity-dependent phenotypes, such as the DAergic phenotype. In the adult brain, the OB and hippocampus are the two principal sites of neurogenesis and GABA also regulates progenitor proliferation, migration, dendritic outgrowth and synaptogenesis in the hippocampus. However, unlike the current study in the OB, the ability of GABA to modulate differentiation of specific hippocampal interneuron phenotypes has not been reported.

A second novel finding in the current study is the molecular inter-relationship between the co-expressed GABAergic and DAergic phenotypes in OB interneurons. Since a majority of OB glomerular layer interneurons express GABA (Kosaka et al. 1998; Parrish-Aungst et al. 2007), determining whether other neuronal phenotypes are also coupled to the co-expressed GABAergic phenotype is of considerable significance. In view of established findings indicating that adult-derived OB interneurons are essential for new odor memory consolidation (reviewed in Lledo et al. 2006), the results of this current study make it tempting to speculate that GABA can modulate neural network plasticity by regulating differentiation of specific molecular phenotypes of OB interneurons.

Supplementary Material

Supplemental Figure


Grant information: supported by NIH R01DC008955


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