To determine if the increase in the number of neurons observed in the inferior colliculus (IC) of the adult genetically epilepsy-prone rat (GEPR) as compared to the Sprague-Dawley rat was present in the young GEPRs prior to the time at which seizure activity commences, brains from both types of rats 4-10 days of age were studied. A statistically significant increase in the numbers of small neurons occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found in the GEPR as compared to the Sprague-Dawley rat and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compensatory to the seizure activity, but is genetically programmed. © 1985

Kim, HL

Ribak, CE

Roberts, RC

To determine if the increase in the number of neurons observed in the inferior colliculus (IC) of the adult genetically epilepsy-prone rat (GEPR) as compared to the Sprague-Dawley rat was present in the young GEPRs prior to the time at which seizure activity commences, brains from both types of rats 4-10 days of age were studied. A statistically significant increase in the numbers of small neurons occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found in the GEPR as compared to the Sprague-Dawley rat and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compensatory to the seizure activity, but is genetically programmed

Roberts, Rosalinda C

Kim, Howard L

Ribak, Charles E

eScholarship - University of California

UC IrvineUC Irvine Previously Published WorksTitleIncreased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone ratPermalinkhttps://escholarship.org/uc/item/2v68v7kdJournalBrain Research, 23(2)ISSN1385-299XAuthorsRoberts, Rosalinda CKim, Howard LRibak, Charles EPublication Date1985-12-01DOI10.1016/0165-3806(85)90050-1Copyright InformationThis work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaDevelopmental Brain Research, 23 (1985) 277-281 277 Elsevier B R D 61) 11)5 Short Communications Increased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone rat ROSALINDA C. ROBERTS, HOWARD L. KIM and CHARLES E. RIBAK Department of Anatomy, University of California, Irvine, CA 92717 (U.S.A.) (Accepted August 13th, 1985) Key words: GABAergic neuron - -  inferior colliculus - -  audiogenic seizure - -  development To determine if the increase in the number of neurons observed in the inferior colliculus (IC) of the adult gen&ically epilepsy-prone rat (GEPR) as compared to the Sprague-Dawley rat was present in the young GEPRs prior to the time at which seizure activity com- mences, brains from both types of rats 4-10 days of age were studied. A statistically significant increase in the numbers of small neu- rons occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found in the GEPR as compared to the Sprague-Dawley rat and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compen- satory to the seizure activity, but is genetically programmed. Previous work from our laboratory has shown that the inferior colliculus (IC) of the adult genetically ep- ilepsy-prone rat (GEPR)  has an increase in the total number  of neurons (mainly the small cells) and an in- crease in the number  of GABAerg ic  neurons 18. This increase of GABAergic  neurons is probably related to the seizure activity in GEPRs  because: (1) other brain regions did not display such a difference 18, (2) the IC is thought to be an important  site for epilepto- genesis 1.8-9.2° and (3) the IC shows electrophysiolog- ical abnormalities2.3. Although previous studies on focal models of epilepsy show a loss of GABAergic  neurons t3-16, genetic models of epilepsy display in- creases in the number  of G A B A  neurons in specific brain regions associated with the analysis of seizure stimuli 12.Is. If the increase in number  of GABAergic  neurons causes an increased inhibition of GABAer -  gic neurons,  then projection neurons would be disin- hibited 12. This hypothesis has been proposed to ex- plain epilepsy in genetic models which exhibit spon- taneous seizures without the introduction of exoge- nous agents into the brain with concomitant  glial scars. The present study was under taken to deter- mine if this increase in cell number  was present in young GEPRs prior to the seizure state because the increased number  of neurons in the adult IC of the GEPR may be a cause of the seizures or a compensa- tory mechanism for the seizure activity. The GEPRs  used in this study were from the same colony used in our previous study IS. The parents of the young GEPRs  were tested and displayed maxi- mal seizures 7. Four GEPRs  and 5 Sprague-Dawley  (SD) rats ranging in age from 4 to 10 days of age were deeply anesthetized with Nembutal  and intracardial- ly perfused with 0.9% saline followed by a 4% para- formaldehyde solution in phosphate buffer (pH 7.4). The brains were dehydrated in alcohol, embedded in paraffin and sectioned in the coronal plane on a ro- tary microtome at a thickness of 10 ~m. Every tenth section throughout the midbrain was stained with cresyl violet and analyzed. The somata of neurons were counted from a repre- Correspondence: R.C. Roberts. Department of Anatomy. University of California, Irvine. CA 92717, U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division) 278 sentative 62,500/~m 2 grid in the ventral  lateral  por-  tion of the central  nucleus of the inferior  colliculus (ICCN) throughout  its ros t rocaudal  extent.  Somata  were classified as small (less than 15 ~tm in d iameter ) ,  medium (15 -25 /~m in d iameter )  or  large (greater  than 25/*m in d iameter )  and the average cell number  in each size group was tabulated,  A grid reticule that was divided into 100 small squares was used to cat- egorize the sizes of neurons.  With  the use of a 40x object ive lens, the length of one side of a square was de te rmined  to be 25 ktm. Thus, neurons  that had a longer d iameter  of half the size of a square or were classified as small; neurons that had diam one-half  to a whole square were classified as n urn; and neurons that had diameters  longer th square were classified as large. The criteria use~ the identif ication of neurons included distinct c plasmic boundaries ,  lucid nuclei and basophil ic plasm and nucleoli.  These features were used tc tinguish neurons from glia. For  example ,  oligo drocytes are small (less than 10/~m) with deepl~ sophilic nuclei. Al though astrocytes are simil~ Figs. I and 2. Photomicrographs of Nissl preparations of the IC. Fig. 1 shows the IC of an 8-10-day-old SD rat at low and high ma$ cations, respectively. Fig. 2 shows age-matched preparations obtained from a GEPR at the same magnifications. The areas of th~ tire IC are similar for both the SD and the GEPR (cf. Figs. la and 2a). However, the GEPR displays a dramatic increase in the nu~ of neuronal somata as compared to the SD (cf. Figs. l b and 2b). A, cerebral aqueduct. Scale bar. 250/~m for l a and 2a and 25/~m fc and 2b. size as the small neurons,  they lack a stained perika- ryal cytoplasm and have stippled nuclei. Quanti ta-  tion of neuron  number  was made by the same individ- ual in a single-blind study where all slides were coded and this code was broken only after all sections were counted. To determine if changes in the number  of neurons were present in other brain regions, neuro- nal somata were also counted from every 10th section in the oculomotor nucleus (cnl l I )  and a portion of the ventral cochlear nucleus in both groups of animals. To determine if the size of the IC was similar in the two types of rats at matched ages, the area of the 1C was determined by tracing the outlines of selected sections on a digitizing tablet connected with an Apple II computer  with an R&M Biometrics soft- ware package. The area of the 1C was calculated rather than the area of the central nucleus alone be- cause the subnuclear boundaries  are unclear at young ages. Data were statistically analyzed using a Student 's  t-test. The Nissl preparations revealed a heterogenous populat ion of small, medium and large neurons in the IC of both groups of animals (Figs. 1 and 2). Howev- er, many more neuronal  somata occurred in the G E P R  as compared to the SD rat at comparable ages. This increase in neuron number  appeared to be due to a selective increase in the small-sized neurons  in both 4 -6  and 8-10 days of age (Figs. lb  and 2b). The density of the Nissl substance appeared to be greater in the G E P R  preparations than in the SD preparations. Therefore,  the somata in the G E P R  material were darker than those in the SD. Quanti tat ive analysis of the preparations con- firmed these visual observations. At  4 days of age there were 116 + 24 small, 20 + 14 medium and 0.8 + 1.1 large neurons  per analyzed grid in the SD (Fig. 3a). In contrast, age matched GEPRs  displayed 180 + 50 small, 18 + 14 medium and 0.1 + 0.3 large neu- rons in the same area (Fig. 3a). Thus,  the 4-day-old GEPRs  had 55% more small neurons  and 40% fewer medium-sized neurons than the SD rats. The large neurons which are infrequent  at this age did not dis- play a significant difference in number  between these two strains of rats. Nevertheless, the G E P R  dis- played more total numbers  of neurons  than the SD rat (198 + 44 vs 147 + 11). The difference in the num- ber of both small and medium-sized neurons  was sta- tistically significant (P < 0.001). 3a 2 0 0  150 100 50 4 - 6  DAYS OF AGE ] SD n=3 • GEPR n=2 • p,c. O0 1 279 3 b  O 2 0 0  l¢ i,u z Q LU 150 Z ~C ~ 1 0 0  ffl Z ~ ~o $ SMALL MEDIUM LARGE <1Sum 15~25um >25um 8 - 1 0  DAYS OF AGE ] SD n-2  • GEPR n=2 p<.O0 1 SMALL  MEDIUM LARGE <1Sum 1 5 - 2 5 u m  >25u rn  Fig. 3. Histograms showing the average number of small, medi- um and large Nissl-stained neurons counted from 62,500 ~m 2 areas in the ICCN, ventrolateral portion, a: at 4-6 days of age the GEPRs display a 55% increase in the number of small neu- rons and a 40% decrease in the number of medium-sized neu- rons relative to the SD rats. There are very few large-sized neu- rons in both these types of animals, b: at 8-10 days of age the GEPRs display a 105% increase in the number of small neu- rons relative to the SD. At this age there are similar numbers of medium-sized and large neurons. Error bars indicate standard deviation; n = number of animals; 17-20 samples were count- ed from each animal. The area of the IC was increased in size in the 8-10-day-old rats as compared to the 4-6-day-old rats. Concomitant  with this increase was a decrease in cell density indicative of the growth and matura- tion of neuronal  processes. At this age, the total number  of neurons in the G E P R  was greater than that of the SD (203 + 27 vs 127 + 27). The data for different size categories were 67 + 22 small, 57 +_ 31 medium and 2.5 +_ 2.8 large neurons for the SD rat and 138 ___ 48 small, 60 _+ 21 medium and 5.3 +_ 5.6 280 I'ABLIZ ! 3,'z,vs/counzs o/twl~rCms m lhe 1(" o/' epi/epm and ncm-epih'/~ti{ rLl[S The data from the present study are summarized under catcgo- ties for 4-6 and 8-10 days of age. For comparison the data from adults I~ are included to show the similarities and differ- ences. 5mall Medium Large tlCHFOtl,~ ?leHroITs HgllrO?lS 4-6-days-old SD rats 116.7 _+ 24.5 3(}.4 _+ 14.0 (}.8 _+ 1.2 GEPRs 180.3_+50.6 18.4_+ 14.2 0.i _+ (}.3 Statistics ¢ -  7.61 t = 3.89 z = 3.26 d r -  82 df = 82 dr= 82 P < 0.001 P < 0.001 8-10-days-old SD rats 67.2 _+ 22.3 57.4 _+ 31.6 2.5 _+ 2.8 GEPRs 138.3 ± 48.4 60.5 _+ 2(I.9 5.3 _+ 5.6 Statistics t = 8.35 t = 0.50 z = 2.79 df = 74 dr= 74 df = 74 P < 0.001 Adult SD rats 26.6 + 10 .9  15.9+5.1 6.7+2.4 GEPRs 50.8 +_ 18.5 21.3 + 7.8 6.2 + 2.5 Statistics z = 7.00 t - 3.659 t = 0.889 d r -  73 df = 73 df = 73 P < 0.(/01 P < 0.001 large neurons  for the G E P R  (Fig. 3b). These  findings represen t  a 105% increase  in the n u m b e r  of  small  neurons  in the G E P R  as c o m p a r e d  to the SD.  No te  that at this age the n u m b e r  of  the med ium-s i zed  and large neurons  was s imilar  in both  groups  of  animals .  A t  both ages the re  were  no s ignif icant  d i f fe rences  in the numbers  of  neurons  in c h i l l  or  the vent ra l  coch-  lear nucleus  b e t w e e n  the two strains of  rats. These  data  suggest  that  a d e v e l o p m e n t a l  defec t  oc- curs in the gene ra t i on  of  small  neu rons  which results  in an increase in the n u m b e r  of  these  neurons  in the I C C N  of the G E P R .  This  increase  in small  neurons  is present  pr ior  to the t ime at which seizure activity c o m m e n c e s .  T h e r e f o r e ,  small  neurons  are  not  gener -  a ted as a resul t  of  the se izure  act ivi ty  in an a t t empt  to compensa t e  for the increased  activity.  The  s i tuat ion is somewha t  d i f ferent  for  the med ium-s i zed  neurons  because  in compa r i son  to SD rats the 4-day-old  G E P R s  had fewer  neu rons  of  this size, the  10-day-old G E P R s  had s imilar  number s ,  and the adults  dis- played an increase  (see Tab le  I). Such da ta  suggest  that  a d e v e l o p m e n t a l  lag in the growth  of  this size neuron may occur  in fl~e G [ : P R .  l~areu ucurons did not show any d i f fe rence  m n u m b e r  h c l ~ e c n  the.' ~w~ strains. Since the area  of  the 1 (  is similar itl bolh groups of animals ,  lhe increases in total nettr~m and small neuron  number s  obse rved  m the ( I E P R  arc real and not simply due to an equal  amoun l  ()f nctl- rons concen t r a t ed  into a smal ler  sized s t ruc l t t r c  Previous  work  has r e p o r t e d  that:  ( 1 ) most  G A B A -  ergic neurons  in the IC are small a l though some  are med ium and a few are large t~ i<, and (2t increased numbers  of G A B A e r g i c  neurons  occur  in the IC of the G E P R  and the m a j o r  increase occurs  m the small  neuronsiS. Since the increase of  small ( i A B A e r g i c  neurons  was so grea t ,  it was ref lec ted  m a grea te r  pe rcen tage  of  the total  n u m b e r  of  neurons  being G A B A e r g i c  in the adult  G E P R  as c o m p a r e d  to the SD and suggested that the defect  may be preferent ia l  for the G A B A e r g i c  systern. The  present  study did not  examine  g lu tamic  acid deca rboxy lase  ( G A D )  im- munocy tochemica l  p repa ra t ions  in these ) o u n g  G E P R s  because  G A D  is not  p resen t  at de tec tab le  levels in this s t ruc ture  at such young  ages (R ibak .  un- publ ished observa t ions) .  H o w e v e r ,  the increase in the n u m b e r  of  small G A B A e r g i c  neurons  in the adult  G E P R  suggests that  the increased  number s  of  small  neurons  obse rved  in the young,  p rese izurc  G E I ' R s  is probably  cor re la ted  with an increase m G A B A  neu- rons. H o w  is this defec t  re la ted  to ep i lep togenes i s  in the G E P R ?  O n e  possibil i ty is that  this defec t  results l:rom the no rad rene rg i c  defici t  that  is found in the  brains of  the G E P R  rats 4-:-I°. N o r e p i n e p h r i n e  (NE)  has been  shown to po ten t i a t e  the act ion of  G A B A  in the cerc-  be l lum of  normal  rats, yet N E  is inef fec t ive  in aug- men t ing  G A B A - m e d i a t e d  inhibi t ion in the G E P R  en. Perhaps  the defect  in the N E  system of G E P R s  trig- gers a c o m p e n s a t o r y  increase in the gene ra t i on  of  in- c reased  number s  of  G A B A e r g i c  neurons .  A second possibil i ty,  the one  which wc fawn ~, is that the addi t ional  G A B A e r g i c  neurons  in the IC of G E P R s  may  be inhibi t ing the tonical ly  act ive G A B A e r g i c  neurons ,  the reby  re leas ing exc i ta to ry  p ro jec t ion  neurons  of  thei r  tonic  inhibi t ion.  This  hy- pothes is  was or iginal ly  p roposed  by Pe te r son  et al. te to expla in  how an increase in the n u m b e r  of  G A B A e r g i c  basket  cells in the h ippocampa l  den ta t e  gyrus of  the se izure-sens i t ive  gerbil  may be causing seizure activity. A pre l iminary  ul t ras t ructural  : t na ly -  281 sis of  gerbi l  p r epa ra t i ons  suppor t s  this hypothes i s  be-  cause m o r e  symmet r i c  synapses  a p p e a r  to be  p resen t  on the  s o m a t a  of  baske t  cells in se izure -sens i t ive  ger-  bils It. W e  plan to e x a m i n e  the  IC  of  the  G E P R  to see if m o r e  s y m m e t r i c  synapses  occur  wi th  G A B A e r g i c  neurons  to add fu r the r  suppor t  for  the  dis inhibi t ion hypothes is  in mode l s  of  gene t ic  epi lepsy.  The  authors  wish to thank  Dr .  Phil l ip J o b e  for  his initial supply of  the  G E P R s  whose  offspr ing were  used in this s tudy and Y a s h o d a  Jhu ran i  and M a r g o t  B rundage  for technical  assistance.  This  p ro jec t  was suppor ted  by a Kl ingens te in  Fe l lowship  ( C . E . R . ) ,  N I H  G r a n t  NS-15669 and a grant  f rom the Epi lepsy  Founda t ion  of  A m e r i c a .  1 Chocholova, L., The role of the cerebral cortex in audioge- nic seizure in the rat, Physiol. Bohemoslovenica, 11 (1962) 452-463. 2 Faingold, C.L., Travis, M.A., Jobe, P.C. and Laird, H.E., Abnormalities of the auditory responses of neurons in the inferior colliculus of genetically epilepsy prone rats, Soc. Neurosci. Abstr., 9 (1983) 400. 3 Gehlbach, G. and Faingold, C.L., Audiogenic seizures and effects of GABA and benzodiazepines (BZDs) on inferior colliculus (IC) neurons in the genetically epilepsy prone (GEP) rat, Fed. Proc. Fed. Am. Soc. Exp. Biol., 43 (1984) 569. 4 Jobe, P.C., Dailey, J.W. and Brown, R.D., Effect of Ro-4- 12284 on audiogenic seizure susceptibility and intensity in epilepsy-prone rats, Life Sci., 28 (1981) 2031-2038. 5 Jobe, P.C. and Laird, H.E., Neurotransmitter abnormali- ties as determinants of seizure susceptibility and intensity in the genetic models of epilepsy, Biochem. Pharmacol., 30 (1981) 3137-3144. 6 Jobe, P.C., Laird, H.E., II, Ko, K.H., Ray, R. and Dailey, J.W., Abnormalities in monoamine levels in the central nervous system of the genetically epilepsy prone rat, Epi- lepsia, 23 (1982) 359-366. 7 Jobe, P.C., Picchioni, A.L. and Chin, L., Role of brain NE in audiogenic seizure in the rat, J. Pharmacol. Exp. Ther., 184 (1973) 1-10. 8 Kesner, R.P., Subcortical mechanisms of audiogenic sei- zures in the rat, Exp. Neurol., 15 (1966) 192-205. 9 Koenig, E., The effects of auditory pathway interruption on the incidence of sound induced seizures in rats, J. Comp. Neurol., 108 (1958) 383-392. 10 Laird, H.E., II, Daily, J.W. and Jobe, P.C., Neurotrans- mitter abnormalities in genetically epilepsy prone rodents, Fed. Proc. Fed. Am. Soc. Exp. Biol., 43 (1984) 2505-2509. 11 Peterson, G.M. and Ribak, C.E., Morphological evidence for increased inhibition of basket cells in the dentate gyrus of Mongolian gerbils, Soc. Neurosci. Abstr., 11 (1985) 1321. 12 Peterson, G.M., Ribak, C.E. and Oertel, W.H., Differ- ences in the hippocampal GABAergic system between sei- zure-sensitive and seizure-resistant gerbils, Brain Res., 340 (1985) 384-389. 13 Ribak, C.E., Axon terminals of GABAergic chandelier cells are lost at epileptic foci, Brain Res., 326 (1985) 251-260. 14 Ribak, C.E., Bradburne, R.M. and Harris, A.B., A prefer- ential loss of GABAergic, inhibitory synapses in epileptic foci: a quantitative ultrastructural analysis of monkey neo- cortex, J. Neurosci., 2 (1982) 1725-1735. 15 Ribak, C.E., Harris, A.B., Vaughn, J.E. and Roberts, E., Inhibitory, GABAergic nerve terminals decrease at sites of focal epilepsy, Science, 205 (1979) 211-214. 16 Ribak, C.E. and Reiffenstein, R.J., Selective inhibitory synapse loss in chronic cortical slabs: a morphological basis for epileptic susceptibility, Can. J. Physiol. Pharmacol., 60 (1982) 864- 870. 17 Roberts, R.C., Ribak, C.E., Kitzes, L.M. and Oertel, W.H., Regional distribution of GABAergic neurons and axons terminals in the brainstem auditory nuclei of the ger- bil, Anat. Rec., 211 (1985) 161A. 18 Roberts, R.C., Ribak, C.E. and Oertel, W.H., Increased numbers of GABAergic neurons in the inferior colliculus of an audiogenic model of genetic epilepsy, Brain Res., in press. 19 Vetter, D.E. and Mugnaini, E., Immunocytochemical lo- calization of GABAergic elements in rat inferior colliculus, Soc. Neurosci. Abstr., 10 (1984) 1148. 20 Wada, J.A., Terao, A., White, B. and Jung, E., Inferior colliculus lesion and audiogenic seizure susceptibility, Exp. Neurol., 28 (1970) 326-332. 21 Waterhouse, B.D. and Jobe, P.C., Effects of norepineph- rine and benzodiazepine on amino acid induced responses of cerebellar Purkinje neurons recorded from the genetical- ly epilepsy prone rat, Soc. Neurosci. Abstr., 10 (1984) 409. 

English

Increased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone rat

ROBERTS, RC

KIM, HL

RIBAK, CE

UC IrvineUC Irvine Previously Published WorksTitleINCREASED NUMBERS OF NEURONS OCCUR IN THE INFERIOR COLLICULUS OF THE YOUNG GENETICALLY EPILEPSY-PRONE RATPermalinkhttps://escholarship.org/uc/item/8v4020z1JournalDEVELOPMENTAL BRAIN RESEARCH, 23(2)ISSN0165-3806AuthorsROBERTS, RCKIM, HLRIBAK, CEPublication Date1985-12-01DOI10.1016/0165-3806(85)90050-1Copyright InformationThis work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaDevelopmental Brain Research, 23 (1985) 277-281 277 Elsevier B R D 61) 11)5 Short Communications Increased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone rat ROSALINDA C. ROBERTS, HOWARD L. KIM and CHARLES E. RIBAK Department of Anatomy, University of California, Irvine, CA 92717 (U.S.A.) (Accepted August 13th, 1985) Key words: GABAergic neuron - -  inferior colliculus - -  audiogenic seizure - -  development To determine if the increase in the number of neurons observed in the inferior colliculus (IC) of the adult gen&ically epilepsy-prone rat (GEPR) as compared to the Sprague-Dawley rat was present in the young GEPRs prior to the time at which seizure activity com- mences, brains from both types of rats 4-10 days of age were studied. A statistically significant increase in the numbers of small neu- rons occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found in the GEPR as compared to the Sprague-Dawley rat and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compen- satory to the seizure activity, but is genetically programmed. Previous work from our laboratory has shown that the inferior colliculus (IC) of the adult genetically ep- ilepsy-prone rat (GEPR)  has an increase in the total number  of neurons (mainly the small cells) and an in- crease in the number  of GABAerg ic  neurons 18. This increase of GABAergic  neurons is probably related to the seizure activity in GEPRs  because: (1) other brain regions did not display such a difference 18, (2) the IC is thought to be an important  site for epilepto- genesis 1.8-9.2° and (3) the IC shows electrophysiolog- ical abnormalities2.3. Although previous studies on focal models of epilepsy show a loss of GABAergic  neurons t3-16, genetic models of epilepsy display in- creases in the number  of G A B A  neurons in specific brain regions associated with the analysis of seizure stimuli 12.Is. If the increase in number  of GABAergic  neurons causes an increased inhibition of GABAer -  gic neurons,  then projection neurons would be disin- hibited 12. This hypothesis has been proposed to ex- plain epilepsy in genetic models which exhibit spon- taneous seizures without the introduction of exoge- nous agents into the brain with concomitant  glial scars. The present study was under taken to deter- mine if this increase in cell number  was present in young GEPRs prior to the seizure state because the increased number  of neurons in the adult IC of the GEPR may be a cause of the seizures or a compensa- tory mechanism for the seizure activity. The GEPRs  used in this study were from the same colony used in our previous study IS. The parents of the young GEPRs  were tested and displayed maxi- mal seizures 7. Four GEPRs  and 5 Sprague-Dawley  (SD) rats ranging in age from 4 to 10 days of age were deeply anesthetized with Nembutal  and intracardial- ly perfused with 0.9% saline followed by a 4% para- formaldehyde solution in phosphate buffer (pH 7.4). The brains were dehydrated in alcohol, embedded in paraffin and sectioned in the coronal plane on a ro- tary microtome at a thickness of 10 ~m. Every tenth section throughout the midbrain was stained with cresyl violet and analyzed. The somata of neurons were counted from a repre- Correspondence: R.C. Roberts. Department of Anatomy. University of California, Irvine. CA 92717, U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division) 278 sentative 62,500/~m 2 grid in the ventral  lateral  por-  tion of the central  nucleus of the inferior  colliculus (ICCN) throughout  its ros t rocaudal  extent.  Somata  were classified as small (less than 15 ~tm in d iameter ) ,  medium (15 -25 /~m in d iameter )  or  large (greater  than 25/*m in d iameter )  and the average cell number  in each size group was tabulated,  A grid reticule that was divided into 100 small squares was used to cat- egorize the sizes of neurons.  With  the use of a 40x object ive lens, the length of one side of a square was de te rmined  to be 25 ktm. Thus, neurons  that had a longer d iameter  of half the size of a square or were classified as small; neurons that had diam one-half  to a whole square were classified as n urn; and neurons that had diameters  longer th square were classified as large. The criteria use~ the identif ication of neurons included distinct c plasmic boundaries ,  lucid nuclei and basophil ic plasm and nucleoli.  These features were used tc tinguish neurons from glia. For  example ,  oligo drocytes are small (less than 10/~m) with deepl~ sophilic nuclei. Al though astrocytes are simil~ Figs. I and 2. Photomicrographs of Nissl preparations of the IC. Fig. 1 shows the IC of an 8-10-day-old SD rat at low and high ma$ cations, respectively. Fig. 2 shows age-matched preparations obtained from a GEPR at the same magnifications. The areas of th~ tire IC are similar for both the SD and the GEPR (cf. Figs. la and 2a). However, the GEPR displays a dramatic increase in the nu~ of neuronal somata as compared to the SD (cf. Figs. l b and 2b). A, cerebral aqueduct. Scale bar. 250/~m for l a and 2a and 25/~m fc and 2b. size as the small neurons,  they lack a stained perika- ryal cytoplasm and have stippled nuclei. Quanti ta-  tion of neuron  number  was made by the same individ- ual in a single-blind study where all slides were coded and this code was broken only after all sections were counted. To determine if changes in the number  of neurons were present in other brain regions, neuro- nal somata were also counted from every 10th section in the oculomotor nucleus (cnl l I )  and a portion of the ventral cochlear nucleus in both groups of animals. To determine if the size of the IC was similar in the two types of rats at matched ages, the area of the 1C was determined by tracing the outlines of selected sections on a digitizing tablet connected with an Apple II computer  with an R&M Biometrics soft- ware package. The area of the 1C was calculated rather than the area of the central nucleus alone be- cause the subnuclear boundaries  are unclear at young ages. Data were statistically analyzed using a Student 's  t-test. The Nissl preparations revealed a heterogenous populat ion of small, medium and large neurons in the IC of both groups of animals (Figs. 1 and 2). Howev- er, many more neuronal  somata occurred in the G E P R  as compared to the SD rat at comparable ages. This increase in neuron number  appeared to be due to a selective increase in the small-sized neurons  in both 4 -6  and 8-10 days of age (Figs. lb  and 2b). The density of the Nissl substance appeared to be greater in the G E P R  preparations than in the SD preparations. Therefore,  the somata in the G E P R  material were darker than those in the SD. Quanti tat ive analysis of the preparations con- firmed these visual observations. At  4 days of age there were 116 + 24 small, 20 + 14 medium and 0.8 + 1.1 large neurons  per analyzed grid in the SD (Fig. 3a). In contrast, age matched GEPRs  displayed 180 + 50 small, 18 + 14 medium and 0.1 + 0.3 large neu- rons in the same area (Fig. 3a). Thus,  the 4-day-old GEPRs  had 55% more small neurons  and 40% fewer medium-sized neurons than the SD rats. The large neurons which are infrequent  at this age did not dis- play a significant difference in number  between these two strains of rats. Nevertheless, the G E P R  dis- played more total numbers  of neurons  than the SD rat (198 + 44 vs 147 + 11). The difference in the num- ber of both small and medium-sized neurons  was sta- tistically significant (P < 0.001). 3a 2 0 0  150 100 50 4 - 6  DAYS OF AGE ] SD n=3 • GEPR n=2 • p,c. O0 1 279 3 b  O 2 0 0  l¢ i,u z Q LU 150 Z ~C ~ 1 0 0  ffl Z ~ ~o $ SMALL MEDIUM LARGE <1Sum 15~25um >25um 8 - 1 0  DAYS OF AGE ] SD n-2  • GEPR n=2 p<.O0 1 SMALL  MEDIUM LARGE <1Sum 1 5 - 2 5 u m  >25u rn  Fig. 3. Histograms showing the average number of small, medi- um and large Nissl-stained neurons counted from 62,500 ~m 2 areas in the ICCN, ventrolateral portion, a: at 4-6 days of age the GEPRs display a 55% increase in the number of small neu- rons and a 40% decrease in the number of medium-sized neu- rons relative to the SD rats. There are very few large-sized neu- rons in both these types of animals, b: at 8-10 days of age the GEPRs display a 105% increase in the number of small neu- rons relative to the SD. At this age there are similar numbers of medium-sized and large neurons. Error bars indicate standard deviation; n = number of animals; 17-20 samples were count- ed from each animal. The area of the IC was increased in size in the 8-10-day-old rats as compared to the 4-6-day-old rats. Concomitant  with this increase was a decrease in cell density indicative of the growth and matura- tion of neuronal  processes. At this age, the total number  of neurons in the G E P R  was greater than that of the SD (203 + 27 vs 127 + 27). The data for different size categories were 67 + 22 small, 57 +_ 31 medium and 2.5 +_ 2.8 large neurons for the SD rat and 138 ___ 48 small, 60 _+ 21 medium and 5.3 +_ 5.6 280 I'ABLIZ ! 3,'z,vs/counzs o/twl~rCms m lhe 1(" o/' epi/epm and ncm-epih'/~ti{ rLl[S The data from the present study are summarized under catcgo- ties for 4-6 and 8-10 days of age. For comparison the data from adults I~ are included to show the similarities and differ- ences. 5mall Medium Large tlCHFOtl,~ ?leHroITs HgllrO?lS 4-6-days-old SD rats 116.7 _+ 24.5 3(}.4 _+ 14.0 (}.8 _+ 1.2 GEPRs 180.3_+50.6 18.4_+ 14.2 0.i _+ (}.3 Statistics ¢ -  7.61 t = 3.89 z = 3.26 d r -  82 df = 82 dr= 82 P < 0.001 P < 0.001 8-10-days-old SD rats 67.2 _+ 22.3 57.4 _+ 31.6 2.5 _+ 2.8 GEPRs 138.3 ± 48.4 60.5 _+ 2(I.9 5.3 _+ 5.6 Statistics t = 8.35 t = 0.50 z = 2.79 df = 74 dr= 74 df = 74 P < 0.001 Adult SD rats 26.6 + 10 .9  15.9+5.1 6.7+2.4 GEPRs 50.8 +_ 18.5 21.3 + 7.8 6.2 + 2.5 Statistics z = 7.00 t - 3.659 t = 0.889 d r -  73 df = 73 df = 73 P < 0.(/01 P < 0.001 large neurons  for the G E P R  (Fig. 3b). These  findings represen t  a 105% increase  in the n u m b e r  of  small  neurons  in the G E P R  as c o m p a r e d  to the SD.  No te  that at this age the n u m b e r  of  the med ium-s i zed  and large neurons  was s imilar  in both  groups  of  animals .  A t  both ages the re  were  no s ignif icant  d i f fe rences  in the numbers  of  neurons  in c h i l l  or  the vent ra l  coch-  lear nucleus  b e t w e e n  the two strains of  rats. These  data  suggest  that  a d e v e l o p m e n t a l  defec t  oc- curs in the gene ra t i on  of  small  neu rons  which results  in an increase in the n u m b e r  of  these  neurons  in the I C C N  of the G E P R .  This  increase  in small  neurons  is present  pr ior  to the t ime at which seizure activity c o m m e n c e s .  T h e r e f o r e ,  small  neurons  are  not  gener -  a ted as a resul t  of  the se izure  act ivi ty  in an a t t empt  to compensa t e  for the increased  activity.  The  s i tuat ion is somewha t  d i f ferent  for  the med ium-s i zed  neurons  because  in compa r i son  to SD rats the 4-day-old  G E P R s  had fewer  neu rons  of  this size, the  10-day-old G E P R s  had s imilar  number s ,  and the adults  dis- played an increase  (see Tab le  I). Such da ta  suggest  that  a d e v e l o p m e n t a l  lag in the growth  of  this size neuron may occur  in fl~e G [ : P R .  l~areu ucurons did not show any d i f fe rence  m n u m b e r  h c l ~ e c n  the.' ~w~ strains. Since the area  of  the 1 (  is similar itl bolh groups of animals ,  lhe increases in total nettr~m and small neuron  number s  obse rved  m the ( I E P R  arc real and not simply due to an equal  amoun l  ()f nctl- rons concen t r a t ed  into a smal ler  sized s t ruc l t t r c  Previous  work  has r e p o r t e d  that:  ( 1 ) most  G A B A -  ergic neurons  in the IC are small a l though some  are med ium and a few are large t~ i<, and (2t increased numbers  of G A B A e r g i c  neurons  occur  in the IC of the G E P R  and the m a j o r  increase occurs  m the small  neuronsiS. Since the increase of  small ( i A B A e r g i c  neurons  was so grea t ,  it was ref lec ted  m a grea te r  pe rcen tage  of  the total  n u m b e r  of  neurons  being G A B A e r g i c  in the adult  G E P R  as c o m p a r e d  to the SD and suggested that the defect  may be preferent ia l  for the G A B A e r g i c  systern. The  present  study did not  examine  g lu tamic  acid deca rboxy lase  ( G A D )  im- munocy tochemica l  p repa ra t ions  in these ) o u n g  G E P R s  because  G A D  is not  p resen t  at de tec tab le  levels in this s t ruc ture  at such young  ages (R ibak .  un- publ ished observa t ions) .  H o w e v e r ,  the increase in the n u m b e r  of  small G A B A e r g i c  neurons  in the adult  G E P R  suggests that  the increased  number s  of  small  neurons  obse rved  in the young,  p rese izurc  G E I ' R s  is probably  cor re la ted  with an increase m G A B A  neu- rons. H o w  is this defec t  re la ted  to ep i lep togenes i s  in the G E P R ?  O n e  possibil i ty is that  this defec t  results l:rom the no rad rene rg i c  defici t  that  is found in the  brains of  the G E P R  rats 4-:-I°. N o r e p i n e p h r i n e  (NE)  has been  shown to po ten t i a t e  the act ion of  G A B A  in the cerc-  be l lum of  normal  rats, yet N E  is inef fec t ive  in aug- men t ing  G A B A - m e d i a t e d  inhibi t ion in the G E P R  en. Perhaps  the defect  in the N E  system of G E P R s  trig- gers a c o m p e n s a t o r y  increase in the gene ra t i on  of  in- c reased  number s  of  G A B A e r g i c  neurons .  A second possibil i ty,  the one  which wc fawn ~, is that the addi t ional  G A B A e r g i c  neurons  in the IC of G E P R s  may  be inhibi t ing the tonical ly  act ive G A B A e r g i c  neurons ,  the reby  re leas ing exc i ta to ry  p ro jec t ion  neurons  of  thei r  tonic  inhibi t ion.  This  hy- pothes is  was or iginal ly  p roposed  by Pe te r son  et al. te to expla in  how an increase in the n u m b e r  of  G A B A e r g i c  basket  cells in the h ippocampa l  den ta t e  gyrus of  the se izure-sens i t ive  gerbil  may be causing seizure activity. A pre l iminary  ul t ras t ructural  : t na ly -  281 sis of  gerbi l  p r epa ra t i ons  suppor t s  this hypothes i s  be-  cause m o r e  symmet r i c  synapses  a p p e a r  to be  p resen t  on the  s o m a t a  of  baske t  cells in se izure -sens i t ive  ger-  bils It. W e  plan to e x a m i n e  the  IC  of  the  G E P R  to see if m o r e  s y m m e t r i c  synapses  occur  wi th  G A B A e r g i c  neurons  to add fu r the r  suppor t  for  the  dis inhibi t ion hypothes is  in mode l s  of  gene t ic  epi lepsy.  The  authors  wish to thank  Dr .  Phil l ip J o b e  for  his initial supply of  the  G E P R s  whose  offspr ing were  used in this s tudy and Y a s h o d a  Jhu ran i  and M a r g o t  B rundage  for technical  assistance.  This  p ro jec t  was suppor ted  by a Kl ingens te in  Fe l lowship  ( C . E . R . ) ,  N I H  G r a n t  NS-15669 and a grant  f rom the Epi lepsy  Founda t ion  of  A m e r i c a .  1 Chocholova, L., The role of the cerebral cortex in audioge- nic seizure in the rat, Physiol. Bohemoslovenica, 11 (1962) 452-463. 2 Faingold, C.L., Travis, M.A., Jobe, P.C. and Laird, H.E., Abnormalities of the auditory responses of neurons in the inferior colliculus of genetically epilepsy prone rats, Soc. Neurosci. Abstr., 9 (1983) 400. 3 Gehlbach, G. and Faingold, C.L., Audiogenic seizures and effects of GABA and benzodiazepines (BZDs) on inferior colliculus (IC) neurons in the genetically epilepsy prone (GEP) rat, Fed. Proc. Fed. Am. Soc. Exp. 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INCREASED NUMBERS OF NEURONS OCCUR IN THE INFERIOR COLLICULUS OF THE YOUNG GENETICALLY EPILEPSY-PRONE RAT

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Increased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone rat

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