Brain stimulation reward is integrated by a network of electrically coupled GABA neurons - PubMed (original) (raw)
Brain stimulation reward is integrated by a network of electrically coupled GABA neurons
Matthew B Lassen et al. Brain Res. 2007.
Abstract
The neural substrate of brain stimulation reward (BSR) has eluded identification since its discovery more than a half-century ago. Notwithstanding the difficulties in identifying the neuronal integrator of BSR, the mesocorticolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) of the midbrain has been implicated. We have previously demonstrated that the firing rate of a subpopulation of gamma-aminobutyric acid (GABA) neurons in the VTA increases in anticipation of BSR. We show here that GABA neurons in the VTA, midbrain, hypothalamus, and thalamus of rats express connexin-36 (Cx36) gap junctions (GJs) and couple electrically upon DA application or by stimulation of the internal capsule (IC), which also supports self-stimulation. The threshold for responding for IC self-stimulation was the threshold for electrical coupling between GABA neurons, the degree of responding for IC self-stimulation was proportional to the magnitude of electrical coupling between GABA neurons, and GJ blockers increased the threshold for IC self-stimulation without affecting performance. Thus, a network of electrically coupled GABA neurons in the ventral brain may form the elusive neural integrator of BSR.
Figures
Figure 1. Co-expression of Cx36 and GAD65/67 in the dorsal VTA
(A-C) These are 20X magnification images of Cx36, GAD65/67 and superimposed Cx36/GAD65/67 fluorescent in-situ hybridization (FISH) in the dorsal VTA of mature rats. Cx36 and GAD65/67 were detected with CY3-labeled (red) and FITC-labeled (green) riboprobes, respectively. All images illustrate DAPI nuclear staining in blue. The insets show enlarged views of the area indicated by the dashed square on the 20X images and reveal the co-expression of Cx36/GAD65/67 in dorsal VTA neurons. Scale bars in B corresponds to all images. The scale bar is equal to 50μm. (D) This graph summarizes the FISH analysis of Cx36+, GAD65/67+, Cx36+ and GAD65/67+, and Cx36- and GAD65/67-neurons in horizontal slices from 4 rat brains. Of the total number of neurons in the dorsal VTA, 27% were Cx36+/GAD65/67+, 39% were Cx36+ only, and 34% were Cx36-/GAD65/67-. All neurons that were GAD65/67+ were Cx36+. (E) This image shows a 20X magnification image of the distribution of Cx36 (red) and GAD65/67 (green) immunoreactivity (IR) in the dorsal VTA. The arrows refer to points of overlap of Cx36- and GAD-IR, seen as yellow pixels, indicative of colocalization of the Cx36 and GAD antigens. Their presence as punctae suggests colocalization is in terminals or neuropil rather than in neuronal somata. These data show that the mRNA transcripts result in expression of Cx36 and GAD65/67 protein in the dorsal VTA.
Figure 2. Co-expression of Cx36 and GAD65/67 in structures along the rostrocaudal neuraxis from the ventral tegmental area (VTA) to the reticular thalamic nucleus (RTN)
(A-G) These are 4X and 20X (A’-G’) magnification images of Cx36/GAD65/67 fluorescent in-situ hybridization (FISH) in the ventral VTA, dorsal VTA, substantia nigra (SN), lateral hypothalamus (LH)/anterior hypothalamus (AH), medial preoptic area (MPA)/medial preoptic nucleus (MPO), ventral hippocampus (HPC), and reticular thalamic nucleus (RTN). Cx36 and GAD65/67 were detected with CY3-labeled (red) and FITC-labeled (green) riboprobes, respectively. All images illustrate DAPI nuclear staining in blue. The insets show enlarged views of the area indicated by the dashed square on the 20X images and reveal the co-expression of Cx36/GAD65/67 in each respective area. Scale bars in A,A’ correspond to all images and is equal to 50μm.
Figure 3. Dopamine-sensitive electrically-coupled GABA neurons are localized to the VTA and nearby structures and correlate with areas showing high expression of GAD 65,67 and Cx36
(A) These superimposed traces show unfiltered recordings of a representative VTA GABA neuron spike before (heavy line) and after (fine line) in situ microelectrophoretic application of DA (+20 nA). Note that VTA GABA neuron spike waveforms are characterized by an initial negative-going deflection followed by a small positive-going potential. The duration of the negative-going component of the spike waveform is approximately 100 μsec. Microelectrophoretic application of DA elicited a trailing spike couplet to the waveform. (B) This ratemeter record shows that DA markedly enhances the firing rate of VTA GABA neurons without any diminution in their activation by repetitive DA current. Despite a marked increase in firing rate with microelectrophoretic pulsing of DA current the leading spike appeared to be unaffected by DA. The coupled spike followed the leading spike faithfully even at DA-evoked firing rates approaching 200 Hz. (C) This trace shows the effects of brief, high-frequency (200 Hz, 10 pulses) stimulation of the internal capsule (IC) on the discharge activity of a VTA GABA neuron. VTA GABA neuron spikes not only accompany each IC stimulation pulse, but are elicited for hundreds of msec after the stimulus train has ended, hence the term “IC-evoked post-stimulus spike discharges” or ICPSDs. (D) Recording sites associated with spikes that were coupled by DA and elicited ICPSDs were subsequently labeled by microelectrophoretic application of the anterograde tracer biotinylated dextran amine (BDA), which labels multiple neurons near the pipette. Calibration bar is 50 μm. (E) This Paxinos and Watson coronal plate at 5.8 mm posterior to bregma shows BDA-labeling sites where GABA neurons were sensitive to DA and ICPSDs were elicited (filled circles). Ventral brain areas showing high levels of Cx36 and GAD65/67 co-expression are depicted in the shaded areas. Note the overlap of BDA-labeled sites and co-labeling for Cx36 and GAD65/67 in the dorsal VTA and the substantia nigra pars reticulata (SNr). Abbreviations: IC – internal capsule; ml - medial lemniscus; PBP – parabrachial pigmented nucleus; RN – red nucleus; SNr - Substantia Nigra pars reticulata; VTA – ventral tegmental area.
Figure 4. Coupling of neurons along the rostrocaudal ventral neuraxis at locations demonstrating co-expression of Cx36 and GAD65/67 transcripts
Spike waveforms and IC-evoked post-stimulus spike discharges (ICPSDs) are shown above Paxinos and Watson coronal plates at locations posterior to bregma (−2.8 to −5.3 mm) in 0.5 mm serial sections. Brief, high-frequency (10 pulses, 200 Hz) stimulation elicited ICPSDs at locations along the rostrocaudal neuraxis from the thalamus to the VTA. The traces above show spike waveforms at stereotaxic locations where ICPSDs were elicited. Biotinylated dextran amine (BDA) was iontophoretically applied from the recording pipette at sites producing ICPSDs. Filled circles indicate locations where BDA-labeled cells were detected (see BDA-labeled neurons in Fig. 3D). Shaded areas indicate high density of co-expression of Cx36 and GAD65/67 (see Fig. 2). BDA-labeled cells corresponding to locations where ICPSDs could be elicited were found in areas that also showed high-density co-expression of Cx36 and GAD65/67. The overlap extended from the midbrain through the lateral hypothalamus to the midbrain. Abbreviations: IC - internal capsule; LH - lateral hypothalamus, ml - medial lemniscus; reticular thalamic nucleus (RTN); SNr - Substantia Nigra pars reticulata; VTA – ventral tegmental area; ZI – zona incerta.
Figure 5. Input/output functions of IC-evoked post-stimulus spike discharges (ICPSDs): Monotonic function of pulse number and stimulus intensity
(A) The inset shows a representative extracellular recording of a VTA GABA neuron during high-frequency stimulation (10 pulses, 200 Hz) of the internal capsule (IC). VTA GABA neuron spikes not only accompany each IC stimulation pulse, but are elicited for hundreds of msec after the stimulus train has ended. The peri-stimulus histogram shows the average of 12 IC stimulations producing ICPSDs. (B) These representative peri-stimulus spike histograms demonstrate that VTA GABA neuron ICPSDs increase with increasing pulse number. (C) This graph summarizes the effects of pulse number on ICPSDs. Fitting these data with linear regression analysis reveals a slope of approximately 4, indicating that 4 discharges occur for every pulse. Moreover, a threshold of 4-5 pulses at half-maximum current intensity is needed to induce discharges. (D) This graph summarizes the effects of pulse number and current on VTA GABA neuron ICPSDs. The goodness of linear fit is shown next to each of the pulse # isobars.
Figure 6. IC self-stimulation responding as a function of pulse number
Once rats reached criterion for IC self-stimulation responding (1000 nosepokes/30 min session; 0.56 Hz) at the active nosepoke responding typically stabilized to throughout the 30 min session and between sessions, with little or no responding at the inactive nosepoke hole. (A) These histograms show IC self-stimulation responding for a typical rat with average responding during 3 separate sessions (top, middle, bottom) wherein the IC pulses were changed from 20 pulses to the value indicated in each histogram. At 15 min into the 30 min session the number of stimulus pulses delivered to the IC was switched from 20 to 4 pulses (top; black to grey on the graph), 20 to 20 pulses (middle), and 20 to 40 pulses (bottom). The rate of responding for the 20 to 4 pulse transition transiently decreased and then subsided markedly, for the 20 to 20 pulse transition produced a mild increase in responding, and for the 20 to 40 pulse transition moderately decreased responding (~ 50%). (B) This graph shows the rate of IC self-stimulation responding as a function of pulse number. Values are expressed as the grand average across 10 rats of the ratio of the average rate of responding after the transition from 20 pulses to the number on the abscissa (second half of the session) vs the average rate of responding with 20 pulses (first half of the session). Compared to the response rate at 20 pulse IC self-stimulation, responding was characterized by an inverted U-shaped curve with threshold at 4-5 pulses, variable responding from 5-10 pulses, and progressive decreases in responding from 20 to 80 pulses.
Figure 7. IC self-stimulation responding as a function of coupling between VTA GABA neurons
(A) The data in Fig. 6B for IC self-stimulation responding as a function of pulse number is expressed here as a function of total current density (in microCoulombs) delivered in a self-stimulation session. Total current density was the product of IC self-stimulation responding times the pulse duration times the current. Note that the threshold remains at 4 pulses, but responding saturates at pulse numbers above 40. (B) This graph shows the relationship between IC self-stimulation current density and the number of VTA GABA neuron ICPSDs produced by a given pulse number. The goodness of linear fit is shown next to the plot, and the equation describing the line is superimposed on the data. There is a strong correlation between VTA GABA neurons ICPSDs and the total current current density delivered during IC self-stimulation.
Figure 8. Gap junction blockers raise the threshold for IC self-stimulation responding
Six rats were trained to perform IC self-stimulation to criterion on 20 IC pulses. To study the effects of the gap junction (GJ) blockers quinidine and mefloquine on threshold for responding we systematically decreased the pulse number within a session for a set number of responses at each pulse level. (A) This graph shows the effects of intraperitoneal administration of the GJ blocker quinidine vs saline on the pulse threshold for IC self-stimulation. Quinidine (20 mg/kg) was administered 10 min before the session. Quinidine significantly decreased overall responding without affecting initial performance for 20 IC pulses at the onset of the session (data not shown--see text). Note that the IC pulse threshold for IC self-stimulation responding was raised by quinidine. (B) This graph shows the effects of intraperitoneal administration of the Cx36 GJ blocker mefloquine vs DMSO vehicle on the pulse threshold for IC self-stimulation. Mefloquine (30 mg/kg) was administered 24 hr before the session. Mefloquine decreased overall responding without affecting initial performance for 20 IC pulses at the onset of the session (data not shown-see text). Note that the IC pulse threshold for IC self-stimulation responding was raised by mefloquine.
References
- Allison DW, et al. Connexin-36 gap junctions mediate electrical coupling between ventral tegmental area GABA neurons. Synapse. 2006;60:20–31. - PubMed
- Connors BW, Long MA. Electrical synapses in the mammalian brain. Annu Rev Neurosci. 2004;27:393–418. - PubMed
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