GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice - PubMed (original) (raw)

GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice

D J Spergel et al. J Neurosci. 1999.

Abstract

Mice were generated expressing green fluorescent protein (GFP) under the control of the gonadotropin-releasing hormone (GnRH) promoter. Green fluorescence was observed in, and restricted to, GnRH-immunopositive neuronal somata in the olfactory bulb, ganglion terminale, septal nuclei, diagonal band of Broca (DBB), preoptic area (POA), and caudal hypothalamus, as well as GnRH neuronal dendrites and axons, including axon terminals in the median eminence and organum vasculosum of the lamina terminalis (OVLT). Whole-cell recordings from GFP-expressing GnRH neurons in the OVLT-POA-DBB region revealed a firing pattern among GFP-expressing GnRH neurons distinct from that of nonfluorescent neurons. Nucleated patches of GFP-expressing GnRH neurons exhibited pronounced responses to fast application of GABA and smaller responses to L-glutamate and AMPA. One-fifth of the nucleated patches responded to NMDA. The GABA-A, AMPA, and NMDA receptor channels on GnRH neurons mediating these responses may play a role in the modulation of GnRH secretory oscillations.

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Figures

Fig. 1.

GFP reporter gene and GFP-expressing neurons in live 300 μm brain slices from GnRH-GFP transgenic mice.A, GFP reporter gene used to generate GnRH-GFP transgenic mice. Restriction sites for cloning (Av, AvrII; H3, _Hin_dIII; X,_Xho_I; N, Not_I;Ba, Bam_HI; Ns, NsiI) and regulatory elements of the minigene [SV40 SD/SA, SV40 splice donor/splice acceptor intron (Zolotukhin et al., 1996);SV40 polyA, SV40 polyadenylation signal] are indicated.B, GFP-expressing neurons in the POA of a coronal slice from a postnatal day 25 (P25) male GnRH-GFP mouse. The dark band in the middle of this image and in_C is the third ventricle (3V).C, GFP-expressing axon terminals in the median eminence from the same mouse as in B. D, GFP-expressing neurons in the DBB of a sagittal slice from a P21 male GnRH-GFP mouse. E, GFP-expressing neurons in the ganglion terminale (GT) of a sagittal slice from a P45 male GnRH-GFP mouse. This mouse and the one from which the images in Figure 6_A were obtained came from a different GnRH-GFP founder line than the mice from which the other images presented here were obtained.

Fig. 2.

Comparison of GFP and GnRH expression in GnRH-GFP transgenic mice. A, GFP-expressing neurons, numbered_1–11_, near the DBB-POA border at the level of the OVLT from the same mouse as in Figure 1_A_ but 300 μm more rostral. The optic chiasm is missing because it detached from the rest of the tissue during slicing. B, Same slice as in_A_ after fixation, which produced additional fluorescence, and after mounting, which flattened the tissue and thereby changed the relative positions of the neurons. Scales in_B–D_ are the same as in A.C, Same slice as in B after immunostaining for GnRH. GnRH-immunopositive neurons are numbered 1–19. The gray levels in this panel and in Figure 3_D_ have been inverted to aid the reader in visualizing the GnRH immunostained neurons, which appear as_dark spots_. Note that all fluorescent neurons in_B_ are GnRH-immunopositive in C and that the number of GnRH-immunopositive neurons is larger than the number of fluorescent neurons. D, Same slice as in_A_ after biocytin labeling of neuron 1.

Fig. 3.

Post hoc identification of GFP-expressing neurons used in physiological recordings.A, High-magnification fluorescence image of GFP-expressing neurons 1 and 2 in Figure2_A_. B, IR-DIC image of_neurons 1_, 2, and 12 in Figure 2_C_. Scales in B–D are the same as in A. C, High-magnification fluorescence image of neuron 1 after biocytin labeling.D, High-magnification fluorescence image of_neurons 1_, 2, and 12 after GnRH immunostaining. Neurons 1 and _2_fluoresce and contain GnRH, whereas neuron 12 contains GnRH but does not fluoresce.

Fig. 4.

Comparison of spontaneous action potentials in GFP-expressing GnRH neurons with those in neighboring nonfluorescent, non-GnRH hypothalamic neurons. A, Fluorescence (left) and IR-DIC (right) images of a GFP-expressing GnRH neuron (1) and a neighboring nonfluorescent neuron (2) during a whole-cell patch-clamp recording of action potentials in the GnRH neuron. The neurons were in the DBB of a sagittal brain slice from a P21 female GnRH-GFP mouse. B, Spontaneous action potentials with long afterhyperpolarizations in GFP-expressing GnRH neurons in the POA of a P44 male GnRH-GFP mouse (left) and in the DBB of a P19 male GnRH-GFP mouse (right). Calibrations in this panel and in C are the same. C, Six shapes of spontaneous action potential in nonfluorescent neurons neighboring GFP-expressing GnRH neurons. Top left, Action potentials in a nonfluorescent neuron neighboring the GnRH neuron whose action potential is shown in the left trace_of B. These action potentials are characterized by a brief afterhyperpolarization, followed by a slow return to the resting potential. Action potentials of this shape were observed in 15 of 26, or 58% of, nonfluorescent neurons. Each of the other action potential shapes shown below was seen in ≤15% of nonfluorescent neurons. Top right, Action potentials in a nonfluorescent neuron neighboring the GnRH neuron whose action potentials are shown in the right trace of_B. In this case, the brief afterhyperpolarization was followed by a fast return to the resting membrane potential.Middle left, Action potentials with a biphasic afterhyperpolarization in a nonfluorescent neuron in the DBB of a 6-month-old female GnRH-GFP mouse. Middle right, Action potentials with little or no afterhyperpolarization in a nonfluorescent neuron in the POA of a P23 female GnRH-GFP mouse. Bottom left, Action potential with an afterhyperpolarization, followed by an afterdepolarization, in a nonfluorescent neuron in the POA of a P42 male GnRH-GFP mouse. Bottom right, Large action potential, followed by a small action potential and afterdepolarization but no afterhyperpolarization, in a nonfluorescent neuron in the DBB of a P15 male GnRH-GFP mouse.

Fig. 5.

Responses of a GnRH neuron to hyperpolarizing and depolarizing current and voltage pulses. A, Passive and active responses to 1 sec current pulses of −50, −40, −30, −20, −10, 0, 10, and 20 pA of a GFP-expressing GnRH neuron in the DBB of the 6-month-old female GnRH-GFP mouse from which the recording in Figure 4_H_ was obtained. B, Maximum firing evoked by a 1 sec depolarizing current pulse of 40 pA in the GnRH neuron whose responses are shown in A.C, High-resolution recording of an action potential evoked by a 20 msec depolarizing current pulse of 40 pA in the same GnRH neuron. Resting, threshold, peak, half-amplitude, and afterhyperpolarization potentials are indicated. D, Voltage-gated currents in the same GnRH neuron in response to 10 msec voltage pulses from a _V_h of −60 mV to test potentials of −140, −120, −100, −80, −60, −40, −20, 0, 20, 40, 60, and 80 mV.

Fig. 6.

GABA-activated currents in GnRH neurons.A, Fluorescence (left) and IR-DIC (right) images of a nucleated patch of a GFP-expressing GnRH neuron from the DBB of a P47 male GnRH-GFP mouse.B, GABA-evoked currents in a nucleated patch of a GFP-expressing GnRH neuron in the POA of a 6-month-old female GnRH-GFP mouse. GABA (1 m

) was applied every 5 sec for 50 msec at_V_h values of −100, −80, −60, −40, −20, 0, 20, 40, 60, and 80 mV. In this and subsequent traces, patches were initially held at −60 mV and then stepped to a new_V_h before agonist and/or antagonist application. Stepping to depolarized test potentials resulted in the outward voltage-gated currents seen before the agonist and/or antagonist response. The voltage-gated currents usually decayed to a steady state by the time of agonist and/or antagonist application. Capacitive currents at the beginning and end of the test pulse are also shown. Scales for these traces and the bottom traces of D are the same. C, Current–voltage relationship of the peak responses in_B_. D, Partial inhibition by bicuculline (Bic) of the response to 1 m

GABA (bottom traces) and full inhibition by bicuculline of the response to 10 μ

GABA (top traces) at_V_h of −100 mV. The patch whose responses are displayed in the bottom traces was from a GnRH neuron in the DBB of a 2-month-old female GnRH-GFP mouse. The patch whose responses are shown in the top traces was from a GnRH neuron in the DBB of a P17 female GnRH-GFP mouse. Capacitive current traces have been blanked.

Fig. 7.

Glutamate-activated currents in GnRH neurons.A, Current responses and current–voltage relationship of the peak responses to 1 m

glutamate (Glu). Arrow points to the responses. The patch is the same as that whose responses to 1 m

GABA are shown in Figure 6_B_. B, Responses to increasing concentrations of glutamate at_V_h of −100 mV. Note the difference in scale compared with that in A. The patch was from a GnRH neuron in the DBB of the same mouse whose responses are shown in_A_. Scale is the same in this panel and in_C_ and D. C, Inhibition of the fast desensitizing component of the glutamate response by NBQX (top traces) and responses to AMPA (middle trace) and kainate (KA; bottom trace), all at V_h of −100 mV. The NBQX-sensitive glutamate responses were from a GnRH neuron in the DBB of a P17 female GnRH-GFP mouse. The AMPA and kainate responses were from a GnRH neuron in the DBB of a 6-month-old female GnRH-GFP mouse.D, Nondesensitizing single-channel responses to glutamate in the absence (top left) and presence (top right) of a cocktail of NBQX and AP-5 at_V_h values of −60, −80, and −100 mV; current–voltage relationship of the single-channel responses (bottom left); and a response to NMDA at_V_h of −100 mV (bottom right). The slope of the current–voltage relationship of the single-channel events (bottom left), which is equivalent to the single-channel conductance (γ), was estimated to be ∼70 pS. The single-channel responses to glutamate (top left and_right) were from a GnRH neuron neighboring the GnRH neuron whose NBQX-sensitive responses are shown in C. Response to NMDA and inhibition by AP-5 (bottom right traces) in a patch from a GnRH neuron in the DBB of a 2-month-old male GnRH-GFP mouse.

Fig. 8.

GABA- and glutamate-activated channels in nucleated patches of nonfluorescent, non-GnRH hypothalamic neurons.A, GABA (1 m

)evoked currents in a nucleated patch from a nonfluorescent hypothalamic neuron neighboring the GFP-expressing neuron from which the nucleated patch responses in Figure 6_A_ were obtained. Scales are the same in_A_ and B. B, Responses to 1 m

glutamate of the neuron whose GABA responses are shown in A. C, Current–voltage relationships for the peak responses to GABA and glutamate in A and_B_. D, Partial inhibition by 50 μ

bicuculline of the response to 1 m

GABA (bottom traces) and complete inhibition by 100 μ

bicuculline of the response to 10 μ

GABA (top traces) at V_h of −100 mV. The patches were from neurons neighboring the GFP-expressing GnRH neurons whose bicuculline-sensitive GABA responses are shown in Figure 6_D. E, Complete inhibition by a cocktail of NBQX and AP-5 of the glutamate response at_V_h of −100 mV. The patch was from the same neuron whose responses are illustrated in the bottom traces of D. F, Inhibition by NBQX of the fast desensitizing component (top traces) and by AP-5 of the nondesensitizing component (bottom traces) of the glutamate response. The patch was from a nonfluorescent neuron in the POA of the same mouse from which the responses of a GnRH neuron (Fig. 7_C_, top traces) were obtained.

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GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice - PubMed (original) (raw)