Neurons in the ventral striatopallidal complex modulate lateral hypothalamic orexin/hypocretin neuron activity: Implications for reward-seeking

Reviewer #1 (Public Review):

Summary:

Using fiber photometry, Mitchell et al. report that the calcium activity of lateral hypothalamic orexin neurons increases during the approach to a food pellet in a manner that depends on the metabolic state and begins to return to baseline prior to and during food consumption. This activity is also enhanced during the approach to palatable food relative to a standard chow pellet. They also present ex vivo electrophysiological evidence that GABAergic neurons in the ventral pallidum and lateral nucleus accumbens shell, but not medial nucleus accumbens shell, provide predominantly inhibitory, monosynaptic input onto lateral hypothalamic neurons. Overall, most claims are well supported by the data, though the electrophysiology analysis is somewhat limited and some information that could inform interpretation of the data is lacking.

Strengths:

(1) The fiber photometry recordings make use of an isosbestic control, and the signals were aligned using linear regression after baseline correction and calculation of robust z-scores.

(2) The fiber photometry analyses are based on animal averages, rather than trial-based averages, which can result in Type 1 errors without appropriate measures to account for the influence of the subject.

(3) Monosynaptic currents from GABAergic inputs from the ventral pallidal and lateral shell are identified by the remaining current in the presence of tetrodotoxin (TTX) and 4-aminopyridine (4-AP).

Weaknesses:

(1) The data are not discussed in the context of the prior literature on ventral pallidal GABAergic inputs to the lateral hypothalamus (such as Prasad et al. 2020, JNeurosci) and it is not clear whether these patterns of monosynaptic inhibitory inputs are specific to orexin neurons.

(2) The paper does not address whether there are synaptic inputs from non-GABAergic ventral pallidum neurons, though very recent work suggests that ventral pallidal projections to the lateral hypothalamus may be enriched with glutamatergic RNA markers relative to other projections (Bernet et al. 2024, JNeurosci). Some statements in the manuscript refer to ventral pallidal inputs in general, despite the use of cell-type specific expression in VGAT-cre mice.

(3) The statistical analysis of the electrophysiology data is limited and does not appear to account for the lack of independence for cells recorded from the same mouse.

Reviewer #2 (Public Review):

Summary:

Mitchell & Mohammadkhani et al. used an Orexin-Cre mouse line with a Cre-dependent GCaMP virus to perform lateral hypothalamic (LH) Ca2+ fiber photometry recordings in mice during the approach to food under various metabolic and saliency conditions. They also used a Vgat-Cre mouse line with Cre-dependent ChR2 in various regions of the ventral striatopallidal (VSP) complex in combination with an Orexin promoter-driven reporter virus labeling Orx-LH neurons to assess electrophysiological connectivity of inhibitory/excitatory inputs from VSP to Orx-LH. Overall, authors note that Orx-LH Ca2+ activation occurs during approach to food (but not consumption of food), and that VSP->Orx-LH connectivity is primarily monosynaptic and inhibitory, although this varies across subregions, with some monosynaptic excitatory input as well. While their methods and analyses are technically sound and the manuscript is clearly written and presented, the further knowledge gained over previous work is rather incremental and does not produce a substantial shift in the current existing framework.

Strengths:

Cell type specificity of OX/HT recordings is confirmed by post-hoc immunostaining, both for fiber photometry and electrophysiological connectivity. This is an important strength given the contentious history of cell specificity in various transgenic OX/HT mouse lines.

Clearly implicating metabolic state and food saliency as factors impacting OX/HT activity dynamics is a strength, and linking the influence of ghrelin receptor signaling is relatively novel.

Weaknesses:

In fiber photometry traces, OX/HT activity begins increasing 2-3 seconds prior to the food approach (Figures 1F and 1G), requiring an explanation. One possibility is that mice may be detecting odorant cues indicative of food prior to the physical approach.

Figure 1F - the authors' interpretation that OX/HT activity doesn't actually decrease during consumption, but simply "trends toward baseline" is complicated by the fact that the authors shaded 20s-30s intervals labeled "eating". Mice do not typically consume food for 20-30s nonstop. Mice typically consume for ~1-5 seconds, then they take a break, then they resume.

The authors state in the Discussion "... the reduction in OX/HT cell activity was more closely correlated with the termination of approach behavior" (rather than with eating per se). However, in many cases, mice begin consuming food immediately after approaching it, so it is puzzling that there is an activity reduction following the approach, but not an activity reduction upon consumption. In other words, the cessation of approach and the beginning of consumption are often tightly linked together in rapid sequence.

Figure 2E - the single polysynaptic oIPSC appears to have the same/similar latency as many of the Monosynaptic oIPSCs. Close proximity of consecutive oIPSCs may affect the analysis of amplitude and latency. For example, in representative traces of Figure 2C, it is unlikely to get an accurate measure of the second oIPSC.

The comparison of apparent connectivity differences between VP vs. mNAcSh vs. lNAcSh is limited by appropriate anatomical quantification and demonstration. When using a Vgat-Cre mouse line and targeting the VSP, there is the potential for massive viral spread across the entire Nucleus accumbens/VP/SI/BNST area.

How do the electrophysiological properties of OX/HT neurons (and VSP inputs) change across metabolic/saliency states? For example, under High Fat Diet, chronic Food Restriction, and chronic Ghrelin. This seems to be the fundamental question that the authors are working toward, but it is not resolved with the current data set.

Potential Ephys Pitfall: a high Chloride internal solution means that oEPSCs might actually be GABAergic after all. Low Chloride solution, so Cl reversal potential is closer to RMP (or put more Chloride in pipette so it has more depolarized potential than resting- to reverse current mediated by Chloride ions). However, the internal solution used for oEPSCs was calculated to have a Cl reversal potential at ~ -20mV; thus, the Cl-mediated PSCs would be depolarizing when cells were held at -65mV. Did the authors apply any blockers in the bath to confirm that recorded oEPSCs were glutamatergic?

Reviewer #3 (Public Review):

Summary:

Orexin/hypocretin (OX/HT) neurons are implicated in food intake and there is evidence supporting OX/HT neurons' role in reward consumption potentially influenced by animal's metabolic state. Here, Mitchell, Mohammadkhani, et al. use fiber photometry to dissociate OX/HT neurons' role in reward-seeking by contrasting their role in reward consumption. Mice were given normal chow or palatable food in a fed or fasted state. The authors recorded GCAMP signals from OX/HT neurons during food approach and consumption. They observed heightened OX/HT GCAMP signals during the food approach; in contrast, they saw the signals decline during arrival at the food source and during food consumption. In a second set of experiments, the authors investigate upstream circuits that could potentially gate OX/HT neurons. They use optogenetics to directly stimulate inhibitory inputs arriving from either the ventral pallidum, the medial, or the lateral nucleus accumbens shell to OX/HT neurons. They investigated if these circuits impinge monosynaptically or polysynaptically onto OX/HT neurons to assess their functional role in inhibiting these neurons. The authors found that the ventral pallidum and the lateral but not medial nucleus accumbens shell exert inhibitory control over OX/HT neurons.

Strengths:

The manuscript is well-written, employs suitable statistical analyses, and the conclusions are generally supported by the results.

Weaknesses:

Larger group sizes in some instances and causal manipulation of the inhibitory circuits during reward approach vs consumption would enable the authors to make stronger assertions about these circuits' role in gating OX/HT neurons in these behaviors.