The Journal of Neuroscience March 1986, 6(3): 613-619

Stimulation of the Lateral Habenula Inhibits -Containing Neurons in the and of the Rat

Greg R. Christoph, Robert J. Leonzio, and Karen S. Wilcox Central Research and Development Department, E. I. du Pont de Nemours & Company, Experimental Station, Wilmington, Delaware 19898

Neurons in the lateral habenula (LHb) of rats have efferent gation provides physiological evidence about the functional re- projections that terminate in the substantia nigra lationship between the LHb and dopaminergic systems. This (SNC) and ventral tegmental area (VTA), where cell bodies of relationship was studied by electrical stimulation of the LHb dopamine-containing neurons are located. In order to study the during single-unit extracellular recording of dopamine-contain- influence of the habenula on dopaminergic activity, single-cell ing neurons in the SNC and VTA of anesthetized rats. electrophysiological techniques were used to record unit dis- The stria medullaris is the major pathway of habenular af- charge of dopamine-containing neurons in the SNC and VTA ferents, and the fasciculus retroflexus contains all habenular during electrical stimulation of the LHb or adjacent structures. efferents (Herkenham and Nauta, 1977, 1979). Electrolytic le- Dopamine-containing neurons in the SNC and VTA were iden- sions of these fiber bundles permitted determination of which tified by their characteristic spike duration (>2 msec), dis- pathway was necessary for habenular stimulation to affect do- charge rate (2-8 spikes/set), and irregular firing pattern. Anal- paminergic activity. Lesions of the LHb with kainic acid, in a ysis of peristimulus time histograms showed that 85% of SNC concentration reported to selectively destroy neuronal perikarya cells and 91% of VTA neurons were inhibited after single pulse in the habenula (Contestabile and Villani, 1983), were made to stimulation (0.25 mA, 0.1 msec) of the LHb. The mean time specify whether the stimulation effects were conveyed by ha- between stimulation and onset of inhibition was 11 msec (range, benular efferents or by fibers of passage through the habenula 2-22 msec) and mean duration of maximal suppression was 76 (Herkenham and Nauta, 1977; lwahori, 1977). msec (range, 20-250 msec). Stimulation of structures adjacent to the LHb (, lateral , medial dorsal thal- Materials and Methods amus, medial habenula) had little or no effect. Destruction of the fasciculus retroflexus, the fiber pathway that contains most Stimulation and recordingprocedures habenular efferents, blocked the stimulation effects on dopa- Male Sprague-Dawley rats, weighing 250-300 gm, were caged in groups mine-containing neurons. Destruction of the stria medullaris, and maintained on a 12/ 12 hr light-dark cvcle with food and wa+pr...... - .ad . which contains most habenular afferents, did not alter the in- lib. Rats were anesthetized withchloral hydrate (400 mg/kg, i.p.) and hibitory effect of habenular stimulation. Injection of a cytotoxin, placed in a stereotaxic instrument. A concentric bipolar tungsten probe kainic acid, in the LHb 1 week before recording sessions blocked (Rhodes SNE-100) served as a stimulating electrode and was lowered to the LHb at stereotaxic coordinates that correspond to atlas coordi- the inhibitory consequences of habenular stimulation. These ex- nates AP 3.6-4.0, ML 0.5-1.0, and DV 0.9-0.0 (Pellegrino et al.. 1979). periments show that activation of neuronal perikarya in the LHb Glass micropipettes used for recording extracellular single-unit activ- causes orthodromic inhibition of dopamine-containing neurons itv were filled with 2 M NaCl that was saturated with Fast D-----preen or-- in SNC and VTA via the fasciculus retroflexus. contained 2% Pontamine sky blue and had tip diameters of l-2 pm (3.0-4.5 MB at 135 Hz). The microelectrode was positioned dorsal to The habenula of the rat is an epithalamic structure with afferents the recording site at a location corresponding to the following atlas from cortical, limbic, and sources (Greatrex and coordinates: SNC, AP 1.9-2.2, ML 1.8-2.2, DV-2.0; VTA, AP 2.4-2.7, Phillipson, 1982; Herkenham and Nauta, 1977). Efferents from ML 0.4-0.8, DV-1.8. The electrode was lowered with a hydraulic mi- crodrive system and extracellular single-neuron action potentials were the lateral habenula (LHb) project via the fasciculus retroflexus amplified, bandpass-filtered (300-3000 Hz), displayed visually, and to numerous target structures, including the substantia nigra monitored on audio with conventional equipment. Action potentials pars compacta (SNC) and the ventral tegmental area (VTA) triggered a window discriminator whose output was integrated and dis- (Herkenham and Nauta, 1979). The anatomical evidence that played in ratemeter form. A laboratory computer (Medical Systems links the LHb with the SNC and VTA suggests that the LHb Corp.) collected unit data and generated peristimulus time histoarams. may exert an influence on dopamine-containing neurons whose Identification of dopamine-containing neurons was based primarily perikarya are located in the SNC and VTA. With the exception on electronhvsiological characteristics (Grace and Bunney, 1983)- - , Pre-- - - of a report on the ability of lesions of the habenula to increase sumed dopamine-containing neurons had triphasic positive-negative- dopamine utilization in frontal cortex (Lisoprawski et al., 1980) positive signals, and the duration of the positive-negative portion was 2-3 msec.The firing patternwas irregular, sometimes bursting, and the there is no direct evidence that habenular efferents control the spontaneous firing rate was 2-7 spikes/set (mean, 4.6 spikeslsec). In activity of dopamine-containing neurons. The present investi- other studies in our laboratory (unpublished observations), neurons with these electrophysiological characteristics had typical responses (Bunney Received Dec. 17, 1984; revised June 10, 1985; accepted June 11, 1985. et al., 1973) to systemic administration of dopaminergic agonists and We thank Beth A. Burkhart for technical assistance and Yvonne J. King for antagonists. Neurons in the region of the SNC and VTA with biphasic preparation of the manuscript. action potential durations of 1.5 msec or less and firing rates of 1O-22 Correspondence should be addressed to Dr. Christoph. spikes/set were presumed to be non-dopaminergic. Copyright 0 1986 Society for Neuroscience 0270-6474/86/030613-07$02,00/O In all experiments, the spontaneous firing rate of the neuron main-

613 614 Christoph et al. Vol. 6, No. 3, Mar. 1986

Inhibit/excite 2.0 SNC Neurons 1.6

1.6 Cell +LH31C E 300 400 500 is 1.4 Y OJ g 1.2

$ L 1.0 v) ?g 08 i li,l,,,,i!,‘nh~y,,~ ce,, +LH22A Lzl . 5 0.6 500 0 100 200 300 400 $ Time (msec) 0.4 Figure 1. Typical responses of presumed dopamine-containing neu- 0.2 1 rons to electrical stimulation of the lateral habenula. Stimulation pulses 0.0 I I I 1 , I were delivered 100 msec after initiation of each of 100 sweeps. Each Pro-Stim 1 2 3 4 5 sweep was 1500 msec in duration, and only the first 500 msec (250 Peri-Stimulus Epochs bins) of the histogram is shown. 2.0 r;llTA Neurons tained a stable baseline for at least 3 min before stimulation of the LHb 1.6 or adjacent structures. Constant-current stimulation pulses (0.25 mA, 1.6 0.1 msec) were delivered at a frequency of 0.67 Hz. Dopamine-con- 9 taining neurons were tested with variation of pulse current (0.0-l .O mA) is 1.4 and pulse duration (0.1-0.5 msec). Sites in the vicinity of the LHb, 23 including the medial habenula, hippocampus, medial dorsal thalamus, g 1.2 and lateral thalamus, were stimulated in order to compare the effects to those of LHb stimulation. Stimulation of the LHb with 1 min trains a$ 10. of pulses (5-20 Hz) was used to measure the effects of repetitive stim- v) ulation on overall firing rate of dopaminergic neurons in the VTA. 2 0.5 Although the majority of recording sites was ipsilateral to the stimu- b lation site, a group of VTA neurons was tested with stimulation of the 6 0.6 contralateral LHb. ii In most experiments, four to eight neurons were tested in each rat. 0.4 Dye spots marking the tip of the microelectrode were made at the end of the experiment by passing -20 PA DC through the pipette for 15- 0.2 (N=15) 30 min. The rats were perfused intracardially with 0.9% saline, followed ” 0.0 by 10% formalin. The brains were removed and 40-Frn-thick sections I I I I I I Pro-Stim 1 3 4 5 were mounted and stained with cresyl violet. Peri-kimulus Epochs Electrolytic lesions Figure p 2. Peristimulus histograms of dopamine-containing neurons Lesions of the stria medullaris (four rats) and fasciculus retroflexus (four that responded to lateral habenula stimulation (0.25 mA, 0.1 msec, 0.67 rats) were made by passing 0.5 mA anodal DC for 15-20 set through Hz) were divided into epochs. Pre-Stim, O-100 msec prior to the stim- the core of the same type of electrode used for stimulation. Lesion sites ulation pulse; Epochs, I-5, successive 50 msec epochs beginning 20 were histologically confirmed as destroying the stria medullaris at AP msec after the stimulation pulse, that is, 20-70, 70-l 20, 120-l 70, 170- 6.2, ML 0.5, and DV 0.2 and the fasciculus retroflexus at AP 3.3, ML 220, and 220-270 msec poststimulation. The spike events per bin per 0.7, DV - 1.0. In these rats, only SNC neurons were studied, and the epoch were averaged for the two categories of response, Inhibit-Excite and Inhibit Only, that were detected in the substantia nigra pars com- time between lesioning and testing was at least 1 hr. pacta (SNC, top) and the ventral tegmental area (VTA, bottom). The response characteristics of SNC and VTA neurons were essentially the Kainic acid lesions same. Kainic acid (1 ng/nI in 0.9% saline) was injected into the LHb from a micropipette (7-10 pm tip) with a hydraulically coupled pump (WP Instruments) at a rate of 25 nl/min. The volume injected was either 125 nl (five rats) or 250 nl (five rats). Two rats received 250 nl of vehicle. these responsive neurons was 76 msec (range, 20-250 msec), The micropipette remained in position 5 min after the injection and and the mean time between stimulation and onset of suppression was then slowly removed. Stimulation and recording sessions occurred 7-8 d after surgery. Dopamine-containing neurons in both SNC and was 11 msec (range, 2-22 msec). VTA were sampled in each rat. In addition to cresyl violet staining, For the purposes of categorization, a response was counted alternate sections from the kainate-injected rats were processed accord- as inhibitory if the probability of spike events during a post- ing to the silver stain procedure (method II) described by Fink and stimulus epoch 50 msec in duration was reduced by 50% or Heimer (1967). more with respect to a prestimulus epoch. A response was count- ed as excitatory if the probability of firing during a poststimulus Results epoch exceeded the control value by 30% or more. Two major categories of response were observed in both SNC and VTA. Stimulation of LHb and adjacent structures The largest category (64%) showed inhibition followed by ex- Analysis of peristimulus time histograms of unit discharge of citation, whereas most other responsive neurons (24%) were dopamine-containing neurons revealed that 85% of SNC neu- only inhibited. Figure 1 shows typical histogram records for the rons (44/52) and 91% of VTA neurons (50155) were completely two types of response, and Figure 2 displays the averaged data suppressed by electrical stimulation (0.25 mA, 0.1 msec pulse) for successive temporal epochs of all histograms in a given of the LHb. The mean duration of complete suppression for subgroup. There were no statistically significant differences (x2 The Journal of Neuroscience Habenular Stimulation inhibits Dopaminergic Neurons 615

Cell +LH36A

12, 5. e no current e 100 208 300 488 we

18.

LHb

HPC

Figure 3. Stimulation of the lateral L. Thal. habenula inhibits dopamine-contain- ing neurons in the VTA, whereas stim- ulation of adjacent sites does not. Sche- matic of brain section shows sites stimulated. Top four histograms, Re- sults from the same neuron at different times with no stimulation current, stimulation of the lateral habenula LHb (LHb), hippocampus(HPC), or lateral thalamus (15. Thal.). Bottom two his- tograms show a different VTA neuron, comparing the stimulation effects ofthe LHb and medial habenula (Mffb). All MHb histograms consist of 100 sweeps, and ii0 296 300 400 508 the stimulus (0.25 mA, 0.1 msec) was delivered 100 msec after initiation of Time (msec) the sweep. tests) between SNC and VTA dopaminergic neurons with re- ventrolateral and anterior to the LHb, which thereby avoided spect to the proportion of responsive cells or to the type of activation of the fasciculusretroflexus, had no effect on dopa- response(Table 1). In both SNC and VTA, the time between mine-containing neurons (n = 14). Fifteen of 17 (88%) VTA onset of inhibition and recovery to the prestimulus level of neuronstested with stimulation of the contralateral LHb were activity was significantly greater (t tests, all p < 0.001) for the inhibited. Inhibit Only response(SNC = 160 msec, VTA = 180 msec) Thirteen of 40 of the non-dopaminergic neuronsin the SNC than for the Inhibit/Excite type (SNC = 106 msec, VTA = 112 and VTA were inhibited by LHb stimulation, and when inhi- msec). For cells in the Inhibit/Excite subgroup,the mean time bition occurred, it was of shorter duration (< 30 msec)than that between stimulation and peak excitation was similar in SNC observed with dopamine-containing neurons.Nine non-dopa- (170 msec)and VTA (180 msec). minergic neuronshad a slight excitatory responseand 18 were Histological analysisshowed that the tip of the stimulating unresponsive. electrode track was within the LHb in all rats for which LHb stimulation resultsare reported. Dye spotsmarking the location Efects of stimulus trains on jiring rate of dopamine-containingneurons were within the SNC or VTA. The firing rate of dopamine-containing neurons was reduced Electrode tracks on which the dye marking technique was not during 1 min trains of pulsesdelivered to the LHb. The amount usedwere observed to penetrate the SNC and/or VTA. of suppressionwas directly related to stimulation frequency (5- Variation of stimulation pulseparameters with 15 dopamine- containing neurons showed that increasing the pulse duration (0.1-0.5 msec) or the stimulation current (0.0-1.0 mA) in- Table 1. Effects of stimulation of the LHb on dopamine-containing creasedthe duration of inhibition. With a fixed pulseduration neurons (0.1 msec),the minimal current required to elicit an inhibitory responsewas 0.125 mA. Number of neurons (% of column total) In nine rats, recordingofa singleneuron wasmaintained while Response SNC VTA Total varying the placementof the stimulating electrode in LHb, hip- pocampus,and lateral thalamus.Figure 3 showsa representative Inhibit/excite 33 (63%) 35 (64%) 68 (64%) examplein which LHb stimulation causedinhibition ofa neuron Inhibit only 11 (21%) 15 (27%) 26 (24%) in VTA, whereasstimulation of the other sitesdid not. Twelve Other response” 2 (4%) 1 (2%) 3 (3%) VTA neurons were tested with stimulation of the medial ha- No response 6 (12%) 4 (7%) 10 (9%) benula (0.25 mA, 0.1 msec pulse). Two of these neuronswere Total 52 (100%) 55 (100%) 107 (100%) slightly inhibited with subsequentexcitation, three cellsshowed only the excitatory component (Fig. 3), and seven others were = Includes atypical response patterns such as excitation only or excitation imbedded unaffected. Stimulation of the medial dorsal thalamus at a site within a longer inhibitory period. 616 Christoph et al. Vol. 6, No. 3, Mar. 1986

Cell #LHb-stim 01 1 2.0 SNC NEURONS 1.8 -

ii 1.6 - 125111 LESION w +’ 1.4 - z al 1.2 - B 0 1.0 -

zP 0.8 - Y w 0.6 - f y 0.4 - 5 min 0.2 - ( KAINIC ACID = I ng/nl) 6X3 N=43 0.0 I I I I I PRE-STIM I 2 3 4 5 PERI-STIMULUS EPOCHS

2.0 VTA NEURONS I .8

z I.6 % +’ I.4 z * I.2

E I .o 2 E 0.8

z 0.6 10 40 4 20 10

0.2 PERCENT CHANGE IN FIRING RATE (KAINIC ACID=lng/nl) 0.0 i Figure 4. Top, Ratemeter record of dopamine-containing neuron in PRE-STIM I 2 3 4 5 the VTA. Stimulation of the LHb (10 Hz, 0.25 mA, 0.1 msec) at the PERI-STIMULUS EPOCHS times indicated suppressed the overall firing rate of the cell. Bottom, Histogram showing the number of VTA dopaminergic cells that re- Figure 6. Effects of kainic acid lesions of the LHb on the responsive- sponded to habenular stimulation (10 Hz, 0.25 mA, 0.1 msec). The ness of dopaminergic neurons to stimulation of the LHb. Mean spike response categories on the abscissa represent the percentage change in events per bin per epoch were averaged over all cells in a given group and are displayed as a function of peristimulus epochs defined as in firing rate during the 1 min stimulation period. Figure 2. Top, Inhibitory effects of habenular stimulation (0.25 mA, 0.1 msec, 0.67 Hz) on neurons in the SNC were blocked by prior injection 2.0 of either 125 or 250 nl kainic acid into the LHb. Bottom, Dopamine- containing neurons in the I7’A were inhibited by habenular stimulation I .8 in the No Lesiongroup and the 125 nl Lesiongroup but were not t I inhibited in the 250 nl Lesiongroup. 5 1.6 m FR LESION +’ I.4 t 20 Hz, n = 8). All of a group of VTA dopaminergic neurons z (n = 43) were testedwith identical stimulation parameters(0.25 = 1.2 - mA, 0.1 msec pulse, 10 Hz). The firing rate during the stimu- k lation period was expressedas a percentageof the firing rate a 1.0 - immediately preceding the stimulation period. Figure 3 shows r z 0.8 - that firing rate was slowed by more than 20% in 36 of the 43 Y neuronstested. Two cells were excited by more than 20%, and w 0.6 - five cells were not clearly affected. The modal responsewas f approximately 50% reduction in firing rate. lg 0.4 - Antidromic activation 0.2 - Throughout the study, neuronswere examined for evidence of , I I I 0.0 ’ I antidromic activation causedby habenular stimulation. Of 57 PRE-STIM I 2 3 4 5 SNC neuronsand 83 VTA neuronstested in nonlesionedrats, PERI-STIMULUS EPOCHS evidence of antidromic activation was detected in only three Figure 5. Effects of lesions of the principal habenular afferent and efferent fiber bundles on the responsiveness of dopamine-containing c SNC neurons to stimulation of the LHb. Peristimulus time histogram epochs are as defined in Figure 2. The mean events per bin per epoch 2. Electrolytic lesion of the stria medullaris (SM Lesion) did not sub- are shown for three groups of rats. Stimulation of the No Lesion control stantiallyalter the response,whereas the responsewas completely blocked group suppressed neuronal activity during poststimulus epochs 1 and after lesion of the fasciculus retroflexus (FR Lesion). The Journal of Neuroscience Habenular Stimulation Inhibits Dopaminergic Neurons 617

Figure 7. Photomicrographsof (A) an untreatedhabenula and (B) the contra- lateralkainic acid-treated(250 nl) ha- benula.Both have been stained with cresyl violet. Note the lack of neuronal cell bodies and the proliferation of glial cells in the LHb in B. The MHb has not been damaged. Bar, 100 pm. LHb, Lateral habenula; MHb, medial haben- ula; V, lateral ventricle. cells in the VTA. Latency of the antidromic spike (2.5-3.0 msec) was highly reproducible. Only one of these neurons was recorded Kainic acid lesionsof the LHb for a long enough period to confirm the antidromic of The inhibitory effect of habenular stimulation was completely the spike with tests of collision and ability to follow double blocked by the 250 nl injection of kainic acid. None of the pulses separated by 5 msec (data not shown). dopamine-containing neurons in SNC (n = 12) or VTA (n = 23) were inhibited by more than 30% in the 250 nl group. The Fasciculus retrojlexus and stria medullaris lesions 125 nl injection also blocked the inhibitory effect of habenular Peristimulus time histograms of SNC cells in rats with fasciculus stimulation on most SNC neurons;only four of 27 neuronswere retroflexus lesions or stria medullaris lesions were compared to inhibited. VTA neurons,however, were as frequently inhibited those of SNC cells in rats without lesions. Figure 5 shows that in the 125 nl group as in the untreated controls (29/33 and 50/ the fasciculus retroflexus lesions blocked the effect of LHb stim- 55 cells inhibited, respectively). Figure 6 showsthe blocking ulation in 19 of 20 neurons. In rats with stria medullaris lesions, effect of kainic acid on the inhibitory response;it also shows all 20 cells tested were inhibited, and in the untreated control that excitatory responsesoccurred 150-200 msec after stimu- group, 44 of 52 neurons were inhibited. lation even in the absenceof a prior inhibition. This was par- 618 Christoph et al. Vol. 6, No. 3, Mar. 1986

titularly evident in the VTA with rats that received 250 nl of kainic acid. Fourteen of 23 (6 1%)neurons in this group showed the excitatory response.Stimulation of the LHb in the two rats that were injected with vehicle instead of kainic acid caused inhibition in 13 of 15 dopaminergic neurons tested (data not shown). The brains of the rats that received kainic acid or vehicle injections weresectioned and treated with a silver stainin order to visualize tissue degeneration.The area surrounding the in- jection pipette wascharacterized by a zone of argyrophilic ma- terial presumedto be cellular debris (Fink and Heimer, 1967). This zone correspondedto elimination of neuronal perikarya, as demonstrated by cresyl violet histology (Fig. 7), and was confined to the lateral aspectof the LHb with the 125 nl injec- tion. With the 250 nl group, the damagedzone extended into the medial aspectof the LHb but did not include the medial habenula. For the rats in the 250 nl group, the damagedarea included approximately 200-500 pm of adjacentthalamus ven- tral and lateral to the LHb. Argyrophilic material characteristic of fiber degenerationwas densestsurrounding the core of the ipsilateral fasciculusretroflexus. The contralateralfasciculus ret- roflexus and contralateral LHb did not contain detectablede- generation.No retrogradedegeneration was detected in the stria medullaris on either side of the brain at points more than 400- 500 pm anterior to the LHb. Figure 8 showsa representation of the degenerationpattern for one of the rats in the 250 nl group. No degenerationwas detected in either of the two rats that received vehicle insteadof kainic acid.

Discussion The experimentalresults reported hereshow that electrical stim- ulation of the LHb causesinhibition of 85-91% of SNC and VTA neurons that have electrophysiologicalcharacteristics of dopamine-containing neurons. Stimulation of habenular neu- rons at 10 Hz causeda reduction in the firing rate of 84% of VTA dopaminergicneurons tested. The LHb hasdirect bilateral efferent projections to the SNC and VTA (Herkenham and Nau- ta, 1979) and stimulation of either the ipsilateral or contralat- era1 LHb wasequally effective in inhibiting DA-containing neu- G. rons. There is some evidence (Cue110et al., 1978; McGeer et al., 1977) that a subsetof efferents from the medial habenula terminate in the VTA. Stimulation of the medial habenula, however, typically had little, if any, inhibitory effect on dopa- I mm minergic neurons. There were no major differencesbetween dopamine-contain- 1 ing neuronsin SNC and VTA with respectto the effectsof LHb . stimulation. The reasonthat someSNC and VTA neuronsdis- I :;:: played inhibition followed by excitation while otherswere only 0 inhibited is unknown. Both types of responsewere often found on the sameelectrode track with no changein stimulation pa- rameters. The excitatory component is probably not a rebound excitation becauseit was sometimesdetected in the absenceof a prior inhibition, particularly in kainate-treated rats and for a few cells tested with stimulation of the medial habenula. Lesions of the stria medullaristhat precededrecording SNC neurons provided data indicating that this afferent pathway is not required for habenular activation to inhibit dopamine-con- taining neurons.In contrast, lesionsof the fasciculusretroflexus Figure 8. Neuronal degeneration 1 week after injection of 250 nl kainic causedvirtually complete blockade of the ability of habenular acid (1 ng/nl) into the LHb of a rat. A-Z, Line drawings of relevant stimulation to inhibit SNC dopamine-containing neurons.Al- structures and dot representation of the areas of degeneration. Panel A though this outcome demonstratesthat an intact fasciculusret- is most anterior and Z most posterior. The injection site was between panels D and E. Note the absence of degeneration in the stria medularis roflexus is necessaryfor habenular activation to suppressdo- (534 in A) and the dense staining surrounding the core of the fasciculus paminergicactivity, it doesnot prove that LHb efferentswithin retroflexus (FR in F-H). No kainate-induced degeneration occurred in the fiber bundle mediate the neuronal response.Two alternate the medial habenula (MHb) nor in any area contralateral to the side of explanations are possible. First, activation of fibers that are injection. known to passthrough the habenula (Herkenham and Nauta, The Journal of Neuroscience Habenular Stimulation Inhibits Dopaminergic Neurons 619

1979;Iwahori, 1977)could affect dopaminergicneuronal activ- it the ability to integrate limbic, basalganglia, and cortical ac- ity. Second,the fasciculusretroflexus contains some habenular tivity for transmission to midbrain structures. For example, afferents, a subsetof which originates in the VTA (Skagerberg physiological studies(Stem et al., 1979; Wang and Aghajanian, et al., 1984; Swanson, 1982). Indeed, antidromic activation of 1977)and neurochemicalmeasurements (Speciale et al., 1980) a few VTA neuronswas detected in our work, and it is possible suggestthat the habenula is a major link between that antidromic activation of thesehabenular afferents,in con- structures and midbrain -containing neurons. The junction with collateral inhibition, could causewidespread in- present study suggeststhat the habenula also servesas a link hibition of dopamine-containingneurons. If either of thesetwo between forebrain structuresand midbrain dopaminergicneu- alternate explanations were correct, then selective destruction rons. of neuronalperikarya while sparingterminals and fibers of pas- sagein the LHb would not block the responseof dopaminergic References neuronsto LHb stimulation. The experiments with kainic acid Bunney,B. S.,J. R. Walters,R. H. Roth, andG. K. Aghajanian(1973) lesionsof the LHb were performed for this reason. Dopaminergicneurons: Effects of antipsychoticdrugs and amphet- The resultsof the silver stain histology suggestthat the kainic amineon singlecell activity. J. Pharmacol.Exp. Ther. 185: 560-57 1. acid lesion in our study did not destroy terminals or fibers of Contestabile,A., andL. Villani (1983)The use of kainicacid for tracing neuroanatomicalconnections in the septohabenulointerpeduncular passage.If terminals and fibers of passagehad been damaged, systemof the rat. J. Comp.Neurol. 214: 459-469. retrograde degenerationwould have been detected in the stria Cuello,A. C., P. C. Emson,G. Paxino,and T. Jesse1(1978) Substance medullaris-as, for example, wasdetected in our laboratory in P containingand cholinergicprojections from the habenula.Brain rats with electrolytic lesions of the LHb (unpublished obser- Res.149: 4 13-429. vations). Sinceafferents and terminals entering the LHb via the Fink, R. P., andL. Heimer (1967) Two methodsfor selectivesilver stria medullaris were sparedfrom damage, it is likely that ef- impregnationof degeneratingaxons and their synapticendings in the ferentsentering the LHb via the fasciculusretroflexus were also centralnervous system. Brain Res.4: 369-374. spared.Therefore, the degenerationsurrounding the core of the Garland,J. C., and G. J. Mogenson(1983) An electrophysiological fasciculusretroflexus probably representsanterograde degen- study of convergenceof entopeduncularand lateral preoptic inputs eration that occurred asa result of destruction of neuronal peri- on lateralhabenular neurons projecting to the midbrain.Brain Res. 263: 33-4 1. karya in the LHb. The core of the fasciculusretroflexus prin- Grace,A. A., andB. S. Bunney (1983) Intracellularand extracellular cipally containsefferents of the medial habenula,whereas LHb electrophysiologyof nigraldopaminergic neurons. I. Identification efferents are reported to surround the core of the fasciculus andcharacterization. J. Neurosci.10: 301-315. retroflexus (Herkenham and Nauta, 1979). Sincelateral haben- Greatrex,R. M., and0. T. Phillipson(1982) Demonstrationof syn- ula and not medial habenulaneurons were damagedby kainic aptic input from prefrontalcortex to the habenulain the rat. Brain acid, and we only sawdegeneration surrounding the core of the Res.238: 192-197. fasciculusretroflexus, our work confirms theseearlier neuroan- Herkenham,M., andW. J. Nauta (1977) Afferent connectionsof the atomical data. habenularnuclei in the rat. A horseradishperoxidase study with a Since kainate-induced destruction of neuronal perikarya in note on the fiber-of-passageproblem. J. Comp.Neurol. 173: 123- 146. the LHb blocked the inhibitory effectsof LHb stimulation, it is Herkenham,M., and W. Nauta (1979) Efferentconnections of the unlikely that our results can be explained by antidromic acti- habenularnuclei in the rat. J. Comp.Neurol. 187: 19-48. vation and collateral inhibition or by activation of fibers of Iwahori,N. (1977) A golgistudy on the habenularnucleus of the cat. passage.Instead, the data indicate that stimulation of neuronal J. Comp.Neurol. 171: 319-344. perikarya in the LHb causesorthodromic inhibition of dopa- Lisoprawski,A., D. Herve, G. Blanc,J. Glowinski,and J. P. Tassin minergic neuronsvia the fasciculusretroflexus. The reasonthat (1980) Selectiveactivation of the mesocortico-frontaldopaminergic a larger volume (250 vs 125 ml) of kainic acid wasrequired to neuronsinduced by lesionof the habenulain the rat. Brain Res.183: block the inhibitory stimulation effects on VTA neurons may 229-234. McGeer, P. L., E. G. McGeer, and T. Hattorl (1977) Dopamine- be related to the additional damagein the medial aspectof the -GABAneuronal linkages in the extrapyramidaland LHb with the larger volume. Perhaps there is a topographical limbicsystems. Adv. Biochem.Psychopharmacol. 16: 397-402. arrangementwhereby medial LHb neuronsproject to the VTA Pellegrino,L. J., A. S. Pellegrino,and A. J. Cushman(1979) A Ste- and lateral LHb neuronsproject to SNC. reotaxic Atlas of the Rat Brain, Plenum,New York. Although the present evidence indicates that activation of Skagerberg,G., 0. Lindvall, andA. Bjijrklund (1984) Origin, course LHb efferents suppressesthe electrical activity of most dopa- andtermination of the mesohabenulardopamine pathway in the rat. mine-containing neurons, whether this effect truly represents Brain Res.307: 99-108. direct inhibition by monosynaptic inputs is not known. The Speciale,S. G., L. M. Neckers,and R. J. Wyatt (1980) Habenular conduction velocity of LHb efferents as determined by anti- modulationof rapheindoleamine metabolism. Life Sci. 27: 2367- dromic activation studies has been reported to vary between 2372. Stem,W., A. Johnson,J. D. Bronzino,and P. J. Morgane(1979) Effects 0.4 and 8.5 m/set (Garland and Mogenson, 1983). If we esti- of electricalstimulation of the lateralhabenula on single-unitactivity mate the length of the efferent pathway to be 4 mm, the cor- of rapheneurons. Exp. Neurol.65: 326-343. respondingtransit time to the SNC/VTA is between 10 and 0.4 Swanson,L. W. (1982) The projectionof the ventraltegmental area m/set. The mean onsettime of inhibition was 11 msecin our andadjacent regions: A combinedfluorescent retrograde tracer and work, so although it is possiblethat dopamine-containingneu- immunofluorescencestudy in the rat. Brain Res.Bull. 9: 321-353. rons are monosynaptically inhibited, a more complex arrange- Wang,R. Y., andG. K. Aghajanian(1977) Physiologicalevidence for ment involving one or more interneurons is plausible. habenulaas a major link betweenforebrain and midbrainraphe. The anatomical connectionsof the habenulamay confer on Science197: 89-9 1.