Correction: Genetic inhibition of neurotransmission reveals role of glutamatergic input to dopamine neurons in high-effort behavior.

In Figure 1e and f, "F4 control" should be "Cre/tdTomato" and "F4Cre KO" should be "F4Cre/tdTomato". In addition, in the Figure1f legend, the first sentence should end with "(Cre/tdTomato: n = 10, F4Cre/tdTomato: n = 14)".In the 'Materials and Methods' section, under 'Electrophysiology,' the n values for evoked action potential recordings were omitted. The sentence 'For high-frequency stimulus-induced action potentials, the stimulus electrode was placed in the rostral part of VTA and a train of 100 Hz stimuli (1 s) was applied' should end with '(Cre/tdTomato: n=10, F4Cre/tdTomato: n=14).'Later in the same paragraph, in 'For recording evoked EPSCs (Cre/tdTomato, n=13, F4Cre/tdTomato, n=15; AMPA EPSCs were recorded at -70 mV and NMDA EPSCs were recorded at +40 mV)', the phrase 'Cre/tdTomato, n=13, F4Cre/tdTomato, n=15' should be deleted; those n values should have appeared at the end of the later sentence beginning 'Miniature ESPCs...'. The complete, corrected sentence is 'Miniature EPSCs (mEPSCs) were acquired in the presence of 0.5-1 µM TTX and 100 µM picrotoxin and semiautomatically detected by offline analysis using in-house software in Igor Pro (Wavemetrics, Portland, OR, USA) (Cre/tdTomato, n=13, F4Cre/tdTomato, n=15).'Finally, in the 'Materials and Methods' section, third sentence under 'Immunohistochemistry,' information for one TH antibody was omitted. The list of antibodies should end with 'or Millipore MAB5280, 1:1000-1:2000.'

Genetic inhibition of neurotransmission reveals role of glutamatergic input to dopamine neurons in high-effort behavior.

Midbrain dopamine neurons are crucial for many behavioral and cognitive functions. As the major excitatory input, glutamatergic afferents are important for control of the activity and plasticity of dopamine neurons. However, the role of glutamatergic input as a whole onto dopamine neurons remains unclear. Here we developed a mouse line in which glutamatergic inputs onto dopamine neurons are specifically impaired, and utilized this genetic model to directly test the role of glutamatergic inputs in dopamine-related functions. We found that while motor coordination and reward learning were largely unchanged, these animals showed prominent deficits in effort-related behavioral tasks. These results provide genetic evidence that glutamatergic transmission onto dopaminergic neurons underlies incentive motivation, a willingness to exert high levels of effort to obtain reinforcers, and have important implications for understanding the normal function of the midbrain dopamine system.

Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays.

To block expression of NMDA receptor NR1 subunit, we injected into rat hippocampus a Herpes Simplex Virus type 1 derived vector bearing a sequence for NR1 antisense. RT-PCR assays with RNA from hippocampus of animals injected either with NR1 antisense vector, control vector or vehicle, showed an amplification signal compatible with NR1 antisense which could be attributed either to an endogenous NR1 antisense or to an artifact. RT-PCR was performed either with different primers or without primers in the RT, using RNA from different tissues. RNAse protection assay was carried out to characterize the amplified signal nature. Our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue. We considered this basal amplification of a mRNA in a RT-PCR assay as "background amplification". After background subtraction, a significant signal only remained when samples from NR1 antisense vector injected animals were used, demonstrating that this was the only source for NR1 antisense. Background amplification at RT in the absence of primers, can constitute a troubling factor in quantitative nucleic acid determination, leading to major interference, particularly when both sense and antisense sequences are present in the sample. Since RT introduced a significant background signal for every gene analyzed, we propose that RT must be always performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal.

Hippocampal infection with HSV-1-derived vectors expressing an NMDAR1 antisense modifies behavior.

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Gene transfer of NMDAR1 subunit sequences to the rat CNS using herpes simplex virus vectors interfered with habituation.

1. The aim is to study some roles of the hippocampal NMDA receptor, by modifying the expression of the essential NR1 subunit, with temporal and spatial restrictions in the central nervous system (CNS) of the rat. 2. Due to their neurotropism and the size of inserts they can accomodate, herpes simplex virus type-1 (HSV-1) derived amplicon vectors were used to transfer sequences, either in sense (+) or antisense (-) orientations, of the NR1 subunit gene, or of the green fluorescent protein (GFP) gene, into the CNS. 3. Vector expression in cell lines was followed by GFP autofluorescence, immunofluorescence and western blot. 4. The vectors were inoculated into the dorsal hippocampus of adult male Wistar rats, which were evaluated for habituation to an open field, and then, for expression of the transgenes, by autofluorescence and western blot; the expression mainly happened in pyramidal cells of CA1. 5. The animals injected with vectors carrying the NR1(+) transgene showed habituation to the new environment, as also happened with rats injected with vectors carrying only the GFP transgene. 6. In contrast, animals injected with vectors carrying NR1(-) sequence, did not show habituation. This might be retrograde amnesia or disability to record the trace, suggesting that the NR1 subunit in the dorsal hippocampus, is involved in habituation to a new environment. 7. HSV-1 derived amplicon vectors appear to be useful tools to modify endogenous gene expression, at a defined period, in restricted regions of the CNS.