Overexpression of 5-HT_1B Receptor in Dorsal Raphe Nucleus Using Herpes Simplex Virus Gene Transfer Increases Anxiety Behavior after Inescapable Stress

Abstract

5-HT(1B) autoreceptors have been implicated in animal models of stress and are regulated selectively by serotonin-selective reuptake inhibitors such as fluoxetine. These terminal autoreceptors regulate serotonin release from dorsal raphe nucleus (DRN) projections throughout rat forebrain. However, it has not been previously possible to manipulate 5-HT(1B) autoreceptor activity selectively without also changing 5-HT(1B) activity in other neurons mediating different behavioral responses. Therefore, we have developed a viral-mediated gene transfer strategy to express hemagglutinin-tagged 5-HT(1B) and manipulate these autoreceptors in DRN. Green fluorescent protein (GFP) was coexpressed from a separate transcriptional unit on the same amplicon to assist in monitoring infection and expression. We confirmed the expression and biological activity of both transgenic proteins in vitro. When injected directly into DRN using stereotaxic procedure, HA-5-HT(1B) receptors were expressed in serotonergic neurons and translocated to the forebrain. The effect of DRN expression of HA-5-HT(1B) on stress-induced behaviors was compared with control rats that received GFP-only amplicons. There was no change in immobility in the forced swim test. However, HA-5-HT(1B) expression significantly reduced entrances into the central region of an open-field arena after water-restraint stress without altering overall locomotor activity, but not in the absence of stress exposure. HA-5-HT(1B) expression also reduced entries into the open arms of the elevated plus maze after water restraint. Because these tests are sensitive to increases in anxiety-like behavior, our results suggest that overactivity of 5-HT(1B) autoreceptors in DRN neurons may be an important mediator of pathological responses to stressful events.

Increased Expression of 5-HT6 Receptors in the Rat Dorsomedial Striatum Impairs Instrumental Learning

“…Previous work from this lab has demonstrated the potential uses of viral-mediated gene transfer to investigate the functions of other serotonin receptors in discrete brain regions (Clark et al, 2002(Clark et al, , 2004Neumaier et al, 2002;Hoplight et al, 2006). We confirmed that the HAtagged 5-HT 6 receptor functioned identically to wild-type receptor in vitro, and the anatomical pattern of distribution of the transgenic 5-HT 6 receptors appears to closely match the normal neuronal distribution of these receptors in striatum, albeit at higher levels.…”

“…In order to visualize neurons expressing transgenes, the GFP protein was coexpressed along with HA-5-HT 6 receptor from the viral amplicon. GFP expression has been shown to be present throughout the cytosol in infected cells (Clark et al, 2002). To allow direct observation of transgenic 5-HT 6 expression, we 'tagged' the 5-HT 6 receptor with HA to identify 5-HT 6 -overexpressing cells.…”

“…We used a modified version of the herpes simplex virus amplicon that expresses a desired transgene and green fluorescent protein (GFP) under separate transcriptional control that has been previously described (Clark et al, 2002). A hemagglutinin (HA) epitope tag coding sequencing was added to the N-terminus of the rat 5-HT 6 receptor sequence by PCR cloning.…”

“…The HA-5-HT 6 sequence of the pHSV-HA6/GFP amplicon was confirmed in its entirety. The pHSV-HA6/GFP amplicon ( Figure 2a) was packaged into HSV viral particles as previously described (Clark et al, 2002) yielding approximately 1 Â 10 8 infective units/ml.…”

“…We used a viral vector injection method modified for the striatum that has been previously described by this and other labs (Carlezon et al, 2000a, b;Clark et al, 2002;Neumaier et al, 2002). At 4 days after surgery, the rats were started on a food-restricted diet of 20 g of rat chow pellets and ad lib water for 10 days before operant conditioning was started.…”