In this study, we will first to identify and characterize synaptic responses. Secondly, the transmitter mechanism mediating the evoked potential will be discussed. Recently, we found that electrical stimulation of the Dl division evoked a negative field potential in the Dm division.
The amplitudes of field potential are depending on stimulation intensity.
Pairs of stimuli, when delivered at brief inter-pulse intervals (IPI), elicited paired pulse facilitation (PPF). Pharmacological data showed that field potential in the Dm division could be inhibited by application of the AMPA/kainate receptor antagonist, CNQX (5μM), 0.5 mM Ca 2+ and 8.0
mM Mg 2+ and TTX (0.5 μM). In contrast, Mg2+ free aCSF and bicuculline upon synaptic responses and prolonged bursting activity with multiple spikes in the Dm division. These results suggest that both GABAergic and glutamatergic transmission may play important roles in the synaptic plasticity of the zebrafish brain.
Long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission are widespread phenomena expressed at possibly every excitatory synapse in the hippocampus and amygdala. In present study, LTP was induced by high frequency stimulation (HFS) or brief bath application of forskolin. Furthermore, LTD was induced by brief bath application of DHPG. Thus, our results suggest that the intratelencephalic connection between Dl and Dm may play an important role in the synaptic plasticity of the zebrafish brain. It also provides a new electrophysiological
4.2. Detailed discussion
4.2.1. Characterize of Dl-evoked field potentials in the Dm division
According to the observations by Northcutt (Northcutt, 2006), tract-tracing studies revealed that neural connections are formed between these regions via afferent Dl fibers projecting to the Dm in goldfish (Cassius auratus). However, the field potential in Dm evoked by Dl stimulation has not been described before. We hypothesized that the connection between Dl and Dm divisions may bear an important intrinsic
physiological role in synaptic function. In this study, electrophysiological procedures were used to identify and characterize synaptic responses in the Dm region following Dl stimulation in vitro using zebrafish brain preparations. We observed an evoked potential in the Dm division when a single-pulse electrical stimulation was applied to the Dl division, suggesting that the extracellular field potentials are a reliable index of synaptic transmission between the Dl and Dm regions.
The field potential can be subdivided into non-synaptic and synaptic components. The following two discuss indicated that P1 component is non-synaptic and N2 is synaptic: first, the amplitude of N2 was invariant during intensity-response function (I-O curve) and paired pulse stimulation. Second, superfusion of 0.5 mM Ca 2+ and 8.0 mM Mg 2+
reversibly abolished N2 but not P1. However, the complete block of field potentials by 0.5 μM TTX showed that all components are dependent on current flow through sodium channels. That evidence suggests that P1 component consists of extracellular spikes not generated through synapses. Herein, we suggesting that the P1 component can be identified as a fiber volley (FV), because Henze et al. (1997) observed that the electrical stimulation of the hippocampal mossy fibers (MF) results in evoked a positive deflection fiber volley (FV) waveform in area CA3 (Henze et al., 1997).
Data from present study showed that N2 component as a population spike (PS) by the electrical stimulation of Dl division. In addition, data showing that the onset latency of PS is slightly decreased upon increasing stimulus amplitude (see Figure 2-3 and Figure 2-7A), suggesting that the
PS is generated through monosynaptic pathway, which consistent with previous studies (Pennartz et al., 1990, Henze et al., 1997, Lewis and O'Donnell, 2000).
In attempting to identify the transmitter system mediating the Dl evoked synaptic response, the following results are of important: (1) superfusion of Mg2+ free aCSF or application of the GABAA antagonist bicuculline (BIC) significantly enhanced the population spike. (2) 5 μM CNQX, an AMPA receptor antagonist completely abolished the population spike, but did not affected the volley fiber.
In neuron, most of the excitatory synaptic signaling is mediated by glutamatergic neurotransmission. Glutamate receptors can be divided into two major classes: non- NMDA and NMDA receptors.
Non-NMDA receptors, such as AMPA and kainite receptor, mediate rapid excitatory synaptic transmission while NMDA receptors play important roles in neuronal plasticity and development. In zebrafish, glutamatergic neurotransmission has been accumulated in numerous areas of the brain, such as retina (Yazulla and Studholme, 2001) and olfactory bud (Edwards and Michel, 2002, 2003). In this study, the Dm field potential was reversibly blocked by CNQX and enhanced by superfusion of Mg2+ free aCSF, suggesting that Dl-Dm pathway synaptic responses are mediated by the glutamatergic neurotransmission. Collectively, these findings are in line with previous studies that have described the field potential in the posterior telencephalon of the zebrafish (Nam et al., 2004a).
The amino acid γ-aminobutyric acid (GABA) represents one of major inhibitory neurotransmitters in the mammalian central nervous system
that has historically received the most attention in alcohol research. In neuron, GABA is synthesized from glutamic acid by glutamic acid decarboxylase (GAD). Recently, zebrafish has been proposed as a models organism for human disorder diseases, such as alcoholism and seizure.
Therefore, information on the function role of GABA neurotransmitter system in zebrafish brain is becoming valuable. Here, we found that the GABAA receptor-mediated inhibition occurs in Dl-Dm synaptic circuit.
Consistent with previous study, in which induced an epileptiform burst in population spikes recorded in the posterior dorsal telencephalon (Kim et al., 2004). We suggest that telencephalic GABAergic system may involve in epileptic seizure activity in zebrafish.
4.2.2. Long-term potentiation at Dl-Dm synapses
In present study, electrophysiological procedures were used to identify and characterize LTP in the Dm region following Dl stimulation in vitro using zebrafish brain preparations. We show that the tetanization of the Dl region induces a robust and stable NMDA receptor-dependent LTP at Dm division. The N-Methyl-D-aspartate (NMDA) receptor is a special type of ionotropic glutamate receptor. It is widely distributed in mammalian brain areas that are implicated in learning and memory, such as the hippocampus and amygdala. In neurons, the activation of the NMDA receptor stimulates Ca2+ influx, leading to the elevation of postsynaptic Ca2+ levels and the subsequent initiation of certain intracellular signal transduction cascades. The NMDA receptor also plays an important role in synaptic plasticity and learning processes. As
reported in previous studies, the high frequency stimulation of the NMDA receptor could evoke long-term potentiation (LTP) in hippocampal slices, which appeared to be a cellular mechanism underlying synaptic plasticity and, ultimately, learning and memory (Bliss and Collingridge, 1993, Beck et al., 2000). The involvement of the NMDA receptor in cognitive function has been further demonstrated in rodents on the basis of their behavioral consequences following the inactivation of the NMDA receptor using specific antagonists. The intrahippocampal infusion of an NMDA receptor antagonist, AP5, has been demonstrated to cause a significant impairment in the inhibitory avoidance task (Izquierdo et al., 1992, Roesler et al., 1998).
In teleost fish, the telencephalic NMDA receptor plays an important role in LTP formation (Nam et al., 2004b) and in spatial (Gomez et al., 2006) and avoidance learning (Xu et al., 2003). In this study, we found that LTP induction was blocked during a period of DL-AP5 perfusion.
However, upon washout of DL-AP5, LTP could be induced. This suggests that an NMDA receptor-mediated component to long-term synaptic plasticity in the Dl–Dm synapses, that may be involved in regulating learning and memory processes.
The extracellular signal-regulated kinase (ERK) pathway is a vital cascade initiated by the stimulation of the NMDA receptor. NMDA receptor signal transduction is implicated in diverse cellular processes, including cell growth, proliferation, differentiation (Marshall, 1995, Ballif and Blenis, 2001), and learning and memory (Quevedo et al., 2004).
Consequently, it is not surprising to find that the physiological
biochemical roles of ERK are highly conserved among vertebrates (Robinson and Cobb, 1997). For example, the FGF/ERK signaling process has been shown to be involved in the induction and patterning of the telencephalon in both mice and zebrafish (Shinya et al., 2001). Recent studies also have shown that the NMDA receptor-modulated ERK activation is required for fear conditioning in the zebrafish. These findings demonstrate that the ERK signal cascade in the telencephalon of zebrafish may also be closely related to LTP.
As was expected, in the present study, the activation of ERK in the telencephalon was induced at 20 min after application of forskolin (50μM). This is consistent with the finding that activation of PKA by application of forskolin to hippocampal slices results in ERK activation in CA1 region (Kanterewicz et al., 2000). In the present study, we confirmed that cAMP signaling pathway is also required for induction of LTP at Dl-Dm synapse.
4.2.3. Long-term depression at Dl-Dm synapses
We show that pharmacological activation of mGluR5 by application of DHPG could triggers a robust, stable and long-lasting LTD. This is consistent with previous observations that DHPG-induced LTD in hippocampal CA1 synapses (Palmer et al., 1997, Fitzjohn et al., 1999, Schnabel et al., 2001, Tokay et al., 2009). The mGluR5 is constitutively expressed and regulates neuronal ion channel activity such as AMPA receptor. In present study, after 10 min application of 25μM DHPG, the field potential response was significantly reduced and continued to
decline until completely abolished. A recent study showed that DHPG induced depression was associated with the internalization of AMPA receptors, which results in removal of AMPA receptors from the synapse (Xiao et al., 2001). Therefore, here we have shown that adult zebrafish and mammalian synapses may share a common molecular mechanism that regulates LTD induction.
4.3. Physiological significance
According to the observations by Maren and Fanselow (Maren and Fanselow, 1995), the axonal projections from the hippocampus to the amygdala might play an important role in synaptic plasticity and context conditioning in the rat. Currently, the telencephalon region of teleost fish has been revealed as an important component of learning and memory, including spatial memory (Saito and Watanabe, 2006, Duran et al., 2008, Broglio et al., 2010) and emotional memory (Portavella et al., 2004, Portavella and Vargas, 2005). In addition, anatomical and functional studies suggest that the Dm and Dl divisions of the telencephalon are homologues of the mammalian amygdala and hippocampus, respectively (Braford, 1995, Mueller et al., 2011). Thus, identification of the teleost telencephalic synaptic circuit is of great interest since telencephalons is playing a key role in many neurological conditions. In summary, our data shows that extracellular field potentials can be evoked in the Dm division following Dl stimulation in vitro. More specifically, the Dm region exhibited both short- and long-term forms of synaptic plasticity, as well as paired pulse facilitation (PPF), long-term potentiation (LTP) and
long-term depression (LTD). These studies have emphasized the possible neural interaction between the Dl and Dm regions in dorsal telencephalon.
Here, we provide the first evidence that suggests the projection from Dl to Dm divisions in zebrafish could correspond to the similar projection from the hippocampus to amygdala in rodents. Our findings present a new possibility for the role of neural connections between the Dl and Dm regions in the mechanisms of learning and memory. Taken together, this knowledge will facilitate use of zebrafish as a model for neurobiological research and as a model for human neurological disorder.
5. Figures
Figure 2-1
Lateral (A), dorsal (B) and coronal plane (C) views of the adult zebrafish brain. The box shows the coronal section in the telencephalon as illustrated in (C). The dorsal telencephalon of zebrafish is further subdivided along the medial-lateral axis into three regions: the dorsal medial (Dm), dorsal lateral (Dl) and the dorsal posterior (Dp) zones. The sulcus ypsiloniformis (Y) appears as a small indentation between the Dm and Dl zone.
Figure 2-2
The field potentials evoked in the dorsal pallium by stimulation of the lateral division (Dl) of the pallium. (A) The telencephalon was placed over the electrode of an MED probe (single electrode size: 50 μm;
inter-electrode distance: 150 μm) and suitable electrodes (white square) were selected for the stimulating cathode and recording cathode (white open square). The sulcus ypsiloniformis (Y) marked the border between the Dm and Dl divisions. (B) Representative network traces of field potential recorded across the 16 sites following electrical test stimulation through one electrode #29 on the telencephalon slice. The numerical values indicate the number of electrode aligned on the MED64 probe from 1 to 64 (8×8 array).
Figure 2-3
The input-output relationship at the Dl-Dm pathway of telencephalon slices. Mean input-output curve was generated by plotting population spike amplitude (mV) over across a range of input stimulus intensities (μA). Population spike amplitude was calculated as the mean of the amplitude of the two consecutive positive peaks to the maximal negative peak.
Figure 2-4
The P1 components of the locally evoked field potential result from direct activation of axons and neurons, whereas N2 are synaptically mediated.
In A, the components of the field potential are indicated. A-C: N2 was reversibly blocked by application of 0.5 mM Ca 2+ and 8.0 mM Mg 2+
perfusion, while P1 was not affected. C, D: all components of the field potential was abolished by 0.5 μM TTX.
Figure 2-5
Paired pulse facilitation of population spike recordings in the dorsal telencephalon. (A) Following paired pulse stimulation of the Dl division, extracellular population spike in the Dm division were collected using varying interpulse intervals (20, 50, 100, 150, 200 ms). (B) The plot summarizes facilitation of the second potential relative to the first one as a function of the short intervals (<200 ms). The amplitude of the population spike were analyzed and all values are reported as the mean ± SEM from 9 slices. ＊＊p < 0.01.
Figure 2-6
AMPA/kainate receptor antagonists reversibly abolish the synaptic response to Dl stimulation. In this experiment, 5 μM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) reversibly abolished the field potential but did not affect the CAP. Washout time was about 60 minutes for CNQX.
Figure 2-7
Effects of Mg2+ free aCSF and bicuculline (BIC) bath application on population spike from telencephalon slices. (A) Overlay of population spike responses at increasing stimulation intensities. In the following experiment, the Test stimulus intensity was adjusted to a level that produced a population spike of 50% of the maximum response. (B) Mg2+
free aCSF produces an epileptiform burst in population spike recorded from Dm. a: single population spike was induced in normal aCSF. b: the evoked bursting activity during perfusion with Mg2+-free aCSF. c: these effects were reversible after wash with normal aCSF. (C) BIC enhances the population spike and produces an epileptiform burst in population spike recorded from Dm. a: single population spike was induced in normal aCSF. b: the amplitude of the population spike was enhanced and the epileptiform activity appeared following application of 3μM BIC. c:
these effects were partially recovery after 2 hr wash.
Figure 2-8
LTP was induced by application of high frequency stimulation (HFS, arrows) in the dorsal telencephalon. (A) A single experiment, the LTP induced by three trains of HFS treatment in the Dl division. LTP-inducing HFS produced a robust and lasting potentiation for 120 min. (B) The average of experiments from 6 slices was used. Each point represents the mean ± SEM of the population spike amplitude. The waveform, recorded before tetanization (solid line) and 1 h post-tetanus (dashed line).
Figure 2-9
The effect of NMDA receptor antagonist, DL-AP5 on LTP induction.
After 15 min of baseline recording, slices were perfused with 40 μM of DL-AP5 (solid line) for 30 min. The first HFS (arrows) was delivered 20 min after the start of the drug per-fusion. The 2nd HFS was delivered 30 min after the washout the DL-AP5. After HFS, stimulation and recording were paused for 10 min for stabilization of the evoked response. LTP induction was blocked when the first HFS was delivered during the period of AP5 superfusion, but not by 2nd HFS after DL-AP5 was washed out.
Figure 2-10
Long-lasting potentiation of field potential induced by forskolin. (A) After 15 min of baseline recording, slices were perfused with 50μM of forskolin for 15 min. The application of forskolin induced LTP that lasted for at least for 60 min. The application of vehicle alone (0.2% DMSO) had no effect on synaptic transmission. The sample traces taken from experiment at the times indicated. (B) Representative Western blots showing regulation of phosphorylated ERK (P-ERK) levels in telencephalon by forskolin.
Figure 2-11
Brief application of the mGluR agonist DHPG induced LTD at Dl-Dm synapses. After application of 25μM DHPG for 10 min, the PS amplitude was substantially depressed and DHPG leads to a long-lasting depression of synaptic transmission that lasted for at least for 1 h.